Protein Gel Electrophoresis Technical Handbook 114311

Western blotting

Protein gel electrophoresis technical handbook separate transfer detect

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Select precast gel

Comprehensive solutions designed to drive your success

Prepare samples and select buffers

Select the standard

Choose the electrophoresis chamber system and power supply

Run the gel

Stain the gel

Post stain

Contents Electrophoresis overview

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Protein gel electrophoresis is a simple way to separate proteins prior to downstream detection or analysis, and is a critical step in most workflows that isolate, identify, and characterize proteins. We offer a complete array of products to rapid, reliable protein electrophoresis for a variety of applications, whether it is the first or last step in your workflow. Our portfolio of high-quality protein electrophoresis products unites gels, stains, molecular weight markers, running buffers, and blotting products for your experiments.

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Gel selection guide Gels

Prepare samples and select buffers Sample prep kits 26 Buffers and reagents 28 Buffers and reagents selection guide 29

Select the standard Protein ladders 34 Protein standards selection guide 36

Choose the electrophoresis chamber system and power supply Electrophoresis chamber systems 50 Electrophoresis chamber system selection guide 51 Power supplies 58

Run the gel Gel run conditions 59 Troubleshooting tips 60

Stain the gel Protein stains 62 Protein stains selection guides 63, 67, 69, 70 Electrophoretic staining technology 71

Post stain Transfer and detection 74

Appendix Protocol quick reference 76 Ordering information 81

For a complete listing of all available products and more, visit thermofisher.com/separate

For ordering information refer to page XX. For quick reference on the protocol please refer to page XX.

Precast protein gels

Sample preparation and electrophoresis buffers

Protein standards

Electrophoresis chamber systems and power supplies

Electrophoresis run conditions

Protein gel stains

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Select the standard


Run the gel

Stain the gel

Post stain

Electrophoresis matrix Electrophoresis is defined as the Two types of matrices are commonly used in transport of charged molecules electrophoresis—polyacrylamide and agarose. The matrices act as porous media and behave like a molecular sieve. through a solvent by an electric Separation of molecules is dependent upon the gel pore size of the matrix used. Agarose has a large pore size and is ideal field. Electrophoresis is a simple, for separating macromolecules such as nucleic acids and protein complexes. Polyacrylamide has a smaller pore size and is ideal for rapid, and sensitive analytical separating most proteins and smaller nucleic acids. tool for separating proteins and Polyacrylamide gel electrophoresis nucleic acids. Any charged ion or (PAGE) molecule will migrate when placed Polyacrylamide gels are generated by the polymerization of acrylamide monomers. These monomers are crosslinked into in an electric field. Most biological long chains by the addition of bifunctional compounds such as (bis), which react with the free molecules carry a net charge at any N,N,-methylenebisacrylamide functional groups of the chain termini. The concentration of acrylamide and bisacrylamide determines the pore size of the gel. pH other than at their isoelectric The higher the acrylamide concentration, the smaller the pore size, resulting in resolution of lower molecular weight molecules and point and will migrate at a rate vice versa. proportional to their charge density. PAGE allows one to separate proteins for different applications

The mobility of a biological molecule through an electric field will depend on the following factors:

based on: • The acrylamide matrix • Buffer systems

Electrophoresis conditions

Linear vs. gradient gels

The separation of molecules is dependent on the electrophoresis conditions. Electrophoresis can be performed under the following conditions:

Gels that have a single acrylamide percentage are referred to as linear gels, and those with a range are referred to as gradient gels. The advantage of using a gradient gel is that it allows the separation of a broader range of proteins than a linear gel.

Continuous vs. discontinuous gels Researchers occasionally refer to gels as continuous or discontinuous. A continuous gel is a gel that has been formed from a single acrylamide solution in the entire gel cassette. A discontinuous gel is formed from two acrylamide solutions, a small, low-percentage stacking gel where the protein wells reside, and a larger portion of gel that separates the proteins. In the traditional Tris-glycine protein gel system, the proteins are stacked in the stacking gel between the highly mobile leading chloride ions (in the gel buffer) and the slower, trailing glycine ions (in the running buffer). The reason for using the stacking gel is to improve the resolution of the bands in the gel. These stacked protein bands undergo sieving once they reach the separating gel.

Mini vs. midi protein gels Commercial gels are available in two size formats, minigels and midigels. Both gels have similar run lengths, but midigels are wider than minigels, allowing midigels to have more wells or larger wells. The additional wells in the midigels permit more samples or large sample volumes to be loaded onto one gel.

Denaturing conditions Electrophoresis is performed under denaturing conditions using an anionic detergent such as sodium dodecylsulfate (SDS). SDS denatures and unfolds the protein by wrapping around the hydrophobic portions. SDS binds at a ratio of ~1.4 g SDS per gram of protein. The resultant SDS-protein complexes are highly negatively charged and are resolved in the gel based on their size.

Nondenaturing (native) conditions Electrophoresis is performed under nondenaturing (native) conditions using buffer systems that maintain the native protein conformation, subunit interaction, and biological activity. During native electrophoresis, proteins are separated based on their charge to mass ratios.

Reducing conditions Electrophoresis is performed under reducing conditions using reducing agents such as dithiothreitol (DTT), β-mercaptoethanol (β-ME) or tris(2-carboxyethyl)phosphine (TCEP). The reducing agents completely unfold the denatured proteins into their subunits by cleaving the disulfide bonds between cysteine residues.

Buffer systems

• Electrophoresis conditions

Electrophoresis is performed using continuous or discontinuous buffer systems. A continuous buffer system utilizes only one buffer in the gel and running buffer. A discontinuous buffer system utilizes a different gel buffer and running buffer1. This system may also use two gel layers of different pore sizes and different buffer composition (the stacking and separating gel). Electrophoresis using a discontinuous buffer system results in concentration of the sample and higher resolution.

• Field strength • Net charge on the molecule • Size and shape of the molecule • Ionic strength

Did you know? Arne Tiselius won the Nobel Prize in Chemistry for electrophoretic analysis of serum proteins in 1948.

Reference

• Properties of the matrix through which the molecules migrate (e.g., viscosity, pore size)

The acrylamide matrix

1. Ornstein L (1964) Disc electrophoresis. 1. Background and theory. Ann N Y Acad Sci 121:321-349.

Mini Gel Tank



Protein standards



Protein gel stains

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Comparison of discontinuous buffer systems SDS-PAGE utilizes a discontinuous buffer system to concentrate or “stack” samples into a very sharp zone in the stacking gel at the beginning of the run. In a discontinuous buffer system, the primary anion in the gel is different (or discontinuous) from the primary anion in the running buffer. Both the Invitrogen™ NuPAGE™ systems (Bis-Tris and Tris-acetate gels) and the Laemmli (Tris-glycine) system are examples of discontinuous buffer systems and work in a similar fashion. However, the NuPAGE system operates at a lower pH as a result of the proprietary ions that are in the system. In a Tris-glycine system (Figure 1), three ions are primarily involved: •C hloride (–), supplied by the gel buffer, serves as the leading ion because it has the highest attraction to the anode relative to other anions in the system. • Glycine (–), the primary anion provided by the running buffer, serves as the trailing ion, because it is only partially negatively charged and remains behind the more highly charged chloride ions in a charged environment. • Tris base (+), is a common ion present in both the gel and the running buffers. During electrophoresis, the gel and buffer ions in the Tris-glycine system form an operating pH of 9.5 in the separating region of the gel. In the case of the Bis-Tris system (Figure 2), three ions are primarily involved: • Chloride (–) supplied by the gel buffer, serves as the fast-moving leading ion. • MES or MOPS (–) (depending on the running buffer choice) serves as the trailing ion. ∙ MES: 2-(N-morpholino) ethane sulfonic acid ∙ MOPS: 3-(N-morpholino) propane sulfonic acid • Bis-Tris (+) acts as the common ion present in the gel while Tris (+) is provided by the running buffer.

With the Tris-acetate system (Figure 3), three ions are primarily involved: • Acetate (–), the leading ion from the gel buffer • Tricine (–), the trailing ion from the running buffer • Tris (+), the common ion (in both gel and running buffer) This system also operates at a significantly lower pH than the Trisglycine system, resulting in less gel-induced protein modifications. The diagrams below (Figures 1, 2, and 3) summarize the migration differences in the stacking gel of each system.


Select the standard


Run the gel

Stain the gel

Post stain

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Select precast gel High-performance precast protein gels If you are doing standard one-dimensional protein electrophoresis, we have a broad range of solutions to fit your research needs. Our selection of precast gels consists of several different chemistries, well formats, and gel sizes, so you can get the protein separation you need for accurate downstream results. Bolt Bis-Tris Plus gel.

Learn more at thermofisher.com/proteingels GLYCINE (Trailing Ion)

PROTEIN/SDS COMPLEX (Stacked Proteins)

CHLORIDE (Leading Ion)

PROGRESSION OF RUN

Figure 1. The Tris-glycine gel system. • Gel buffer ions are Tris and chloride (pH 8.7) • Running buffer ions are Tris, glycine, and SDS (pH 8.3) • Gel operating pH is 9.5

Common Ion is Tris, present in the gel and running buffers

MES or MOPS (Trailing Ion)


CHLORIDE (Leading Ion)

Figure 2. The Bis-Tris gel system. • Gel buffer ions are Bis-Tris and chloride (pH 6.4) • Running buffer ions are Tris, MES or MOPS, and SDS (pH 7.3) • Gel operating pH is 7.0

Precast gels Popular gel chemistries

Specialty gels

• NuPAGE Bis-Tris gels

• Novex Tricine gels

• NuPAGE Tris-Acetate gels

• NativePAGE gels

• Bolt Bis-Tris Plus gels

• Novex IEF gels

• Novex Tris-Glycine gels

• Novex Zymogram gels • E-PAGE gels

Did you know? Over 45 years ago, Ulrich K. Laemmli first published SDS-PAGE as a method for cleavage analysis of structural proteins in bacteriophage T4.

PROGRESSION OF RUN Common Ion is Bis-tris, present in the gel

Casting your own gels? TRICINE (Trailing Ion)


ACETATE (Leading Ion)

Figure 3. The Tris-acetate gel system. • Gel buffer ions are Tris and acetate (pH 7.0) • Running buffer Ions are Tris, tricine, and SDS (pH 8.3) • Gel operating pH is 8.1

We offer preassembled empty cassettes, buffers, and reagents.

 Learn more at thermofisher.com/gelcastingaccessories

PROGRESSION OF RUN Common Ion is Tris, present in the gel and running buffer

The combination of a lower-pH gel buffer (pH 6.4) and running buffer (pH 7.3–7.7) leads to a significantly lower operating pH (pH 7.0) during electrophoresis, resulting in better sample integrity and gel stability.



Protein standards



Protein gel stains

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Select precast gel

Select the standard

Gel selection guide

Choose the electrophoresis chamber system and power supply chamber system and power supply

Run the gel

Stain the gel

Find the right gel for your research needs based on molecular weight, downstream applications, and throughput requirements.

Molecular weight Low molecular weight proteins and peptides (>2.5 kDa)

High molecular weight proteins (<500 kDa)

Novex Tricine gels

NuPAGE Tris-Acetate gels

High-sensitivity western blotting

Broad-range molecular weight separation

Low throughput

Medium or high throughput

Application

E-PAGE 48-well or 96-well gels

Downstream applications requiring high protein integrity (e.g., mass spectrometry)

Large sample volume for high detection sensitivity

Bolt Bis-Tris Plus gels

NuPAGE Bis-Tris gels






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The most widely used gel system for separating a broad range of proteins by SDS-PAGE is the Laemmli system, which uses Tris-glycine gels comprising a stacking gel component that helps focus the proteins into sharp bands at the beginning of the electrophoretic run and the resolving gel component that separates the proteins based on size. This classic system uses a discontinuous buffer system where the pH and ionic strength of the buffer used for running the gel (Tris, pH 8.3) is different from the buffers used in the stacking gel (Tris, pH 6.8) and resolving gel (Tris, pH 8.8). The highly alkaline operating pH of the Laemmli system may cause band distortion, loss of resolution, or artifact bands. The major causes of poor band resolution with the Laemmli system are:

Novex Tris-Glycine gels

• Hydrolysis of polyacrylamide at the high pH of the resolving gel, resulting in a short shelf life of 8 weeks • Chemical alterations such as deamination and alkylation of proteins due to the high pH of the resolving gel

Native separation

Molecular weight 1st dimension 2nd dimension

Isoelectric point

NativePAGE gels

Novex Tris-Glycine gels with native buffers

Novex IEF gels

ZOOM™ IPG strips

NuPAGE Bis-Tris gels, 2D well

Novex Tris-Glycine gels, 2D well

Novex Tris-Glycine gels, 2D well

Novex Tris-Glycine ZOOM™ gels, IPG well

NuPAGE Bis-Tris gels, 2D well

NuPAGE Bis-Tris ZOOM gels, IPG well

 Find the right mini gel using our interactive gel selection tool at thermofisher.com/minigelselection

For ordering information refer to pages 81–87.

Protease activity Novex Zymogram gels (casein, blue casein, or gelatin substrates)

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Choose the right gel chemistry for your research needs Bis-Tris chemistry vs. Tris-glycine chemistry

Denaturing separation

Coomassie dye or silver staining

Protein gel electrophoresis technical handbook

Post stain

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Figure 4. Protein separation using (A) a Bolt Bis-Tris Plus gel and (B) another manufacturer’s traditional Tris-glycine gel.

Unlike traditional Tris-glycine gels, NuPAGE and Bolt gels are BisTris HCI–buffered (pH 6.4) and have an operating pH of about 7.0. The neutral operating pH of the Bis-Tris systems provides the following advantages over the Laemmli system: • Longer shelf life of 8–12 months due to improved gel stability • Improved protein stability during electrophoresis at neutral pH enabling sharper band resolution and accurate results (Moos et al. 1998) • Complete reduction of disulfides under mild heating conditions (70°C for 10 minutes) and absence of cleavage of Asp-Pro bonds • Reduced state of the proteins maintained during electrophoresis and blotting of the proteins when using Invitrogen™ NuPAGE™ Antioxidant

• Reoxidation of reduced disulfides from cysteine-containing proteins, as the redox state of the gel is not constant • Cleavage of Asp-Pro bonds of proteins when heated at 100°C in Laemmli sample buffer, pH 5.2

Choosing the right gel percentage In general, the size of the molecule being separated should dictate the acrylamide or agarose percentage you choose. Use a lower percentage gel to resolve larger molecules and a higher percentage gel to resolve smaller ones. The exception to this rule is when performing isoelectric focusing. Refer to the gel migration charts throughout this chapter to find the gel best suited for your application. As a general rule, molecules should migrate through about 70% of the length of the gel for the best resolution. When protein molecular weights are wide ranging, or unknown, gradient gels are usually the best choice.



Protein standards

Choosing a well format and gel thickness We offer most polyacrylamide gels in a choice of nine different well formats (17 well, 15 well, 12 well, 10 well, 9 well, 5 well, 1 well, 2D/preparative well, or IPG well). Two thicknesses (1.0 mm and 1.5 mm) are also available for popular gel types. If loading large sample volumes (>30 μL), a thicker gel (1.5 mm) with fewer wells (e.g., 5 well) or a Bolt gel with its higher-capacity wedge wells is more appropriate. When blotting, that proteins will transfer more easily from a 1.0 mm thick gel than from a 1.5 mm thick gel.



Protein gel stains

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Run the gel

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Post stain

Bolt Bis-Tris Plus mini gels

Figure 7. Bolt Bis-Tris Plus gel migration chart. Optimal separation range is shown within the gray areas.

Neutral-pH gel system with a unique wedge well design Invitrogen™ Bolt™ Bis-Tris Plus gels are precast polyacrylamide gels designed for optimal separation of a broad molecular weight range of proteins under denaturing conditions during gel electrophoresis (Figure 6 and 7). These gels help deliver consistent performance with a neutral-pH environment to minimize protein degradation. The unique wedge well design (Figure 5) allows loading of up to 2x more sample volume than other precast gels. Bolt gels are ideal for western blot transfer and analysis along with any other technique where protein integrity is crucial. Bolt Bis-Tris Plus gels offer:

Specifications • Shelf life: ~16 months • Average run time: 35 minutes • Separation range: 0.3–260 kDa

• Better protein resolution—gels are 10% longer, allowing detection of more protein bands than standard mini gels • High lot-to-lot consistency—coefficient of variation (CV) of only 2% for Rf values (migration)

Figure 5. The unique wedge well design of Bolt Bis-Tris Plus gels.

“For one of our projects in the lab, we resolve proteins by electrophoresis to determine the accumulation of ubiquitinated proteins following treatment with a proteasome inhibitor. When we resolved the ubiquitinated proteins using the Tris-glycine gels, we observed a smear. However, when we switched to resolving the ubiquitinated proteins using the Bolt Bis-Tris gels, we were delightfully surprised to observe individual protein bands in place of the smear.” —Susan S., University of Pennsylvania, Philadelphia, US

• Polyacrylamide concentrations: fixed 8%, 10%, and 12%; gradient 4–12% • Gel dimensions: 8 x 8 cm (1 mm thick) • Maximum sample volume per 12-well gel: ~40 μL, or two-thirds of the sample well volume

• Preserved protein integrity—neutral-pH formulation minimizes protein modifications • Superior band quality and band volume— Invitrogen™ Novex™ Bis-Tris Plus chemistry is designed to deliver sharp, straight bands with higher band volume

“The new Bolt system is wonderful. I am still amazed that I can run a PAGE gel in 23 minutes. The entire system is incredibly friendly from the Bolt precast gels with wedged wells for ease of loading to the Mini Gel Tank system. The bands produced from the westerns were sharp and straight. I would and have highly recommended this system to anyone doing protein work.” — Crystal M., Queen’s University, Ontario, Canada

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• High sample volume capacity—wedge well design allows detection of proteins in very dilute samples or measurement of low-abundance proteins

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Figure 6. Bolt Bis-Tris Plus gel electrophoresis. Protein standards and samples were loaded at 10 μL sample volumes in a Bolt 4–12% Bis-Tris Plus Gel. Electrophoresis was performed using the Mini Gel Tank at 200 V (constant). Sharp, straight bands with consistent migration patterns were observed after staining with Invitrogen™ SimplyBlue™ SafeStain. Images were acquired using a flatbed scanner. Lane 1: Invitrogen™ SeeBlue™ Plus2 Prestained Standard; Lane 2: 10 μg E. coli lysate; Lane 3: Invitrogen™ Mark12™ Unstained Standard (blend of 12 purified proteins); Lane 4: 40 μg HeLa cell lysate; Lane 5: 20 μg HeLa cell lysate; Lane 6: 5 μg BSA; Lane 7: 40 μg Jurkat cell lysate; Lane 8: 5 μg GST fusion protein; Lane 9: Invitrogen™ Novex™ Sharp Unstained Protein Standard; Lane 10: 5 μg β-galactosidase.

Did you know? Timothy Updyke and Sheldon Engelhorn filed a patent for the neutral-pH Bis-Tris gel system in 1996.

Recommended products The Invitrogen™ Bolt™ Welcome Pack + iBlot™ 2 System offers a complete protein separation and western blot solution by combining our Mini Gel Tank, Invitrogen™ Bolt™ gels and buffers, SeeBlue Plus2 Prestained Standard, and Invitrogen™ iBlot™ 2 Gel Transfer Device with transfer stacks.

Thermo Scientific Pierce Power Stainer is recommended for fast Coomassie dye staining of Bolt Bis-Tris Plus Gels.

The Bolt Welcome Pack + iBlot 2 System.

 Learn more at thermofisher.com/bolt For ordering information refer to page 81. For quick reference on the protocol please refer to page 76.



Protein standards



Protein gel stains

Select precast gel


Select the standard


Run the gel

Stain the gel


Post stain

NuPAGE gels

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NuPAGE gels A.

Revolutionary high-performance gels referenced in >20,000 publications The Invitrogen™ NuPAGE™ SDS-PAGE gel system is a revolutionary high-performance polyacrylamide gel electrophoresis system that simulates the denaturing conditions of the traditional Laemmli system. NuPAGE™ gels use a unique buffer formulation to maintain a neutral operating pH during electrophoresis, which minimizes protein modifications that can result in poor band resolution. Gels are available in two formulations— Invitrogen™ NuPAGE™ Bis-Tris gels are ideal for separating small to midsize proteins while Invitrogen™ NuPAGE™ Tris-Acetate gels are ideal for separating large proteins (Figure 8). A gel migration chart is shown in Figure 9.

NuPAGE Bis-Tris gels (Denaturing separation)

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NuPAGE Tris-Acetate gels (Denaturing separation)

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NuPAGE Tris-Acetate gels (Native separation)

Specifications • Shelf life: –– NuPAGE Bis-Tris gels: 16 months –– NuPAGE Tris-Acetate gels: 8 months • Average run time: ~35 minutes • Separation range: –– NuPAGE Bis-Tris gels: 1.5–300 kDa –– NuPAGE Tris-Acetate gels: 30–400 kDa

NuPAGE gels are designed for:

• Polyacrylamide concentrations: –– NuPAGE Bis-Tris gels: fixed 8%, 10%, and 12%; gradient 4–12% –– NuPAGE Tris-Acetate gels: fixed 7%; gradient 3–8%

• Superior protein band resolution and stability— neutral-pH environment during electrophoresis minimizes protein modifications

• Gel dimensions: –– Mini: 8 x 8 cm (1 or 1.5 mm thick) –– Midi: 8 x 13 cm (1 mm thick)

• More efficient western blot transfer—neutral pH prevents reoxidation of reduced samples during protein transfer

• Maximum sample volume per 10-well mini gel: 25 µL (1 mm thick); 37 µL (1.5 mm thick)

• Fast sample run times—typically 35–50 minutes • Long product shelf life—stable for 8–16 months

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Figure 8. NuPAGE Bis-Tris and Tris-Acetate gel electrophoresis. Protein standards and samples were loaded at 10 μL sample volumes  th e k in (A) Invitrogen™ NuPAGE™ 4–12% Bis-Tris and Mini Gel Tan (B) Invitrogen™ NuPAGE™ 3–8% Tris-Acetate gels. Electrophoresis was performed using the Mini Gel Tank at 200 V (constant). Sharp, straight bands were observed after staining with SimplyBlue SafeStain. Images were acquired using a flatbed scanner. (A and B) Lane 1: SeeBlue Plus2 Prestained Standard; Lane 2: 10 μg E. coli lysate; Lane 3: Mark12 Unstained Standard (blend of 12 purified proteins); Lane 4: 40 μg HeLa cell lysate; Lane 5: 20 μg HeLa cell lysate; Lane 6: (A) not used (B) 5 µg BSA; Lane 7: 40 μg Jurkat cell lysate; Lane 8: 5 μg GST fusion protein; Lane 9: Novex Sharp Unstained Protein Standard; Lane 10: 5 μg β-galactosidase.

 Learn more at thermofisher.com/nupage For ordering information refer to page 81. For quick reference on the protocol please refer to page 76-77.

Figure 9. Migration patterns achieved in NuPAGE gels. For optimal results, protein bands should migrate within the gray shaded areas. (A) Migration patterns of Invitrogen™ Novex™ Sharp Prestained Protein Standard or Novex Sharp Unstained Protein Standard on NuPAGE Bis-Tris

gels. (B) Migration patterns of HiMark Unstained Protein Standard on NuPAGE Tris-Acetate gels. (C) Migration pattern for Tris-acetate gel native separation is for the Invitrogen™ NativeMark™ Unstained Protein Standard.

Recommended products Invitrogen™ HiMark™ Unstained and Prestained Protein Standards are specifically designed for large protein analysis on NuPAGE Tris-Acetate gels under denaturing conditions. Both standards offer a ready-to-load format and consist of 9 proteins with a size range of 40–500 kDa.

Visualize with Coomassie stain, silver stain, or fluorescent protein stains after electrophoresis (see “Stain the gel”, page 62).


PageRuler, PageRuler Plus, and Spectra Prestained Protein Ladders are recommended for use with NuPAGE Bis-Tris gels for easy molecular weight determination.


Protein standards



Protein gel stains

Select precast gel


Select the standard


Run the gel

Stain the gel


Post stain


A.

Laemmli-based precast gels for high efficiency, reproducibility, and performance

Novex Tris-Glycine gels are: • Individually packaged for convenience • Compatible with most protein standards for accurate size determination • Flexible for use with native or denatured protein samples, with specially formulated buffers for each condition

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Denaturing separation

Native separation††

Tris-Glycine Gels Large proteins* (116–500 kDa) 0%

Invitrogen™ Novex™ Tris-Glycine gels are based on traditional Laemmli protein electrophoresis with minor modifications for maximum performance in the precast format. These gels provide reproducible separation of a wide range of proteins into wellresolved bands (Figure 10). A gel migration chart is shown in Figure 11.

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Specifications

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Mid-size proteins† (20–250 kDa) 8%

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• Shelf life: 1–2 months

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• Run time: ~90 minutes

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Figure 10. Novex Tris-Glycine gel electrophoresis. Protein standards and samples were loaded at 10 μL sample volumes in 4–20% Tris-Glycine gels. Electrophoresis was performed using the Mini Gel Tank at 200 V (constant). Sharp, straight bands were observed after staining with SimplyBlue SafeStain. Images were acquired using a flatbed scanner. Lane 1: SeeBlue Plus2 Prestained Standard; Lane 2: 10 μg E. coli lysate; Lane 3: Mark12 Unstained Standard (blend of 12 purified proteins); Lane 4: 40 μg HeLa cell lysate; Lane 5: 20 μg HeLa cell lysate; Lane 6: 5 μg BSA; Lane 7: 40 μg Jurkat cell lysate; Lane 8: 5 μg GST fusion protein; Lane 9: Novex Sharp Unstained Protein Standard; Lane 10: 5 μg β-galactosidase.

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• Maximum sample volume per well: 25 μL (1 mm thick); 37 μL (1.5 mm thick)

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• Gel dimensions: –– Mini: 8 x 8 cm (1 or 1.5 mm thick) –– Midi: 8 x 13 cm (1 mm thick)

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• Polyacrylamide concentrations: –– Fixed concentrations available from 4% to 18% –– Gradient gels with ranges of 4–12%, 4–20%, 8–16%, and 10–20%

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Figure 11. Migration patterns of protein molecular weight standards in Novex Tris-glycine gels. For optimal results, protein bands should migrate within the gray shaded areas. (A) *Migration patterns of HiMark™ Unstained Protein Standard. † Migration patterns of Novex Sharp PreStained Protein Standard or Novex Sharp Unstained Protein Standard. (B) † † Migration pattern of NativeMARK Unstained Protein Standard.

Recommended products or sample cleanup prior to electrophoresis, we recommend using the F Pierce SDS-PAGE Sample Prep Kit.

Buffers for native proteins: Invitrogen™ Novex™ Tris-Glycine Native Sample Buffer and Novex™ Tris-Glycine Native Running Buffer.

uffers for denatured proteins: Invitrogen™ Novex™ Tris-Glycine SDS B Sample Buffer and Novex™ Tris-Glycine SDS Running Buffer.

PageRuler, PageRuler Plus, and Spectra protein ladders are recommended for molecular weight determination with Novex Tris-Glycine gels.

 Learn more at thermofisher.com/trisglycine For ordering information refer to page 82–83. For quick reference on the protocol please refer to page 77-78.



Protein standards



Protein gel stains

Select precast gel


Select the standard


Run the gel

Stain the gel


Post stain

NativePAGE gels

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NativePAGE gel

Superior resolution of native proteins and protein complexes The Invitrogen™ NativePAGE™ Bis-Tris gel system is based on the blue native polyacrylamide gel electrophoresis (BN PAGE) technique that uses Coomassie G-250 dye as a charge shift molecule that binds to proteins and confers a negative charge without denaturing the proteins (Figure 12). This technique overcomes the limitations of traditional native electrophoresis by providing a near-neutral operating pH and detergent compatibility. The near-neutral (pH 7.5) environment of the NativePAGE system during electrophoresis results in maximum protein and gel matrix stability, enabling better band resolution than other native gel systems. A gel migration chart is shown in Figure 13. The NativePAGE gel system is designed for:

Specifications • Shelf life: 6 months • Average run time: 90 minutes

Did you know?

• Separation range: 15–10,000 kDa

The blue native polyacrylamide gel electrophoresis technique was developed by Hermann Schagger and Gebhard von Jagow in 1991.

• Polyacrylamide concentrations: gradient 3–12% and 4–16% • Gel dimensions: 8 x 8 cm (1 mm thick) • Maximum sample volume per 10-well gel: 25 μL

Is there a higher res pic somewhere? I copied and pasted this from source file.

• A wide resolving range—from 15 kDa to over 10 MDa (Figure 12), regardless of isoelectric point • Neutral-pH separation—the native state of protein complexes is better preserved • Superior performance—higher resolution than Tris-glycine native electrophoresis


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dvantages of the NativePAGE gel system over A the Tris-glycine gel system include:

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Figure 13. NativePAGE gel migration chart. Migration patterns of the NativeMark Unstained Protein Standard on NativePAGE gels are shown.

Figure 12. NativePAGE gel electrophoresis. Two-fold dilution series of protein extracts were run on an Invitrogen™ NativePAGE™ Novex™ 3–12% BisTris Protein Gel using a Mini Gel Tank. Following electrophoresis, the gel was stained with Coomassie dye and imaged using a flatbed scanner. Lanes 1 and 10: blank; Lanes 2 and 6: 5 μL NativeMark Unstained Protein Standard; Lanes 3, 4 and 5: 10, 5, and 2.5 μg spinach chloroplast extract; Lanes 7, 8 and 9: 10, 5, and 2.5 μg bovine mitochondrial extract.

• Reduced vertical streaking—Coomassie G-250 dye binds to nonionic detergent molecules in the sample and carries them in the dye front, ahead of resolving proteins • Better separation of proteins—positively charged proteins with high isoelectric points are converted to proteins with a net negative charge, allowing migration to the anode

Recommended products NativeMark Unstained Protein Standard is recommended for use with native gel chemistries, including our Tris-glycine, Tris-acetate, and NativePAGE gel systems. This standard offers a wide molecular weight range of 20–1,200 kDa, and the 242 kDa β-phycoerythrin band is visible as a red band after electrophoresis for reference (prior to staining). See page 40 for details.

• Minimized protein aggregation—Coomassie G-250 dye binding allows separation of membrane proteins and proteins with exposed hydrophobic areas

 Learn more at thermofisher.com/nativepage For ordering information refer to page 84. For quick reference on the protocol please refer to page 78.



Protein standards



Protein gel stains

Select precast gel


Select the standard


Run the gel

Stain the gel


Post stain

Novex Tricine gels

19

Novex Tricine gel

High-resolution gels for peptide analysis and low molecular weight proteins The Invitrogen™ Novex™ Tricine gel system is a modification of the Tris-glycine system in which tricine replaces glycine in the running buffer. This system uses a discontinuous buffer system specifically designed for the resolution of low molecular weight proteins (Figure 14). Advantages of Novex Tricine gels over Tris-glycine gels include: • Increased resolution of proteins with molecular weights as low as 2 kDa (Figure 15)

Specifications • Shelf life: 1–2 months • Average run time: 90 minutes

• Gel dimensions: 8 x 8 cm (1 mm thick) • Maximum sample volume per 10-well gel: 25 μL

In the traditional Tris-glycine protein gel system, the resolution of smaller proteins (<10 kDa) is hindered by the continuous accumulation of free dodecyl sulfate (DS) ions from the SDS sample and running buffers in the stacking gel, which causes mixing of the DS ions with smaller proteins and results in fuzzy bands and decreased resolution. The mixing also interferes with the fixing and staining of smaller proteins. The Novex Tricine gel system uses a low pH in the gel buffer and sub-

Figure 15. Novex Tricine gel migration chart. For optimal resolution, protein bands should migrate within the shaded areas.






• Minimized protein modification due to the lower pH of the tricine buffering system

How Novex Tricine gels work

Sample preparation is not the only factor that can result in poorly resolved bands. You can minimize protein degradation by using gels with neutral-pH chemistry.

• Polyacrylamide concentrations: fixed 10% and 16%; gradient 10–20%

• Improved compatibility with direct protein sequencing applications after transferring to PVDF membranes

Good to know

Did you know?




18

th e

Mini Gel Tan

k

Figure 14. Novex Tricine gel electrophoresis. Protein standards and samples were loaded at 10 μL sample volumes on Invitrogen™ Novex™ 10–20% Tricine Protein Gels. Electrophoresis was performed using the Mini Gel Tank at 200 V (constant). Sharp, straight bands were observed after staining with SimplyBlue SafeStain. Images were acquired using a flatbed scanner. Lane 1: SeeBlue Plus2 Prestained Standard; Lane 2: 10 μg E. coli lysate; Lane 3: Mark12 Unstained Standard (blend of 12 purified proteins); Lane 4: 40 μg HeLa cell lysate; Lane 5: 20 μg HeLa cell lysate; Lane 6: 5 μg BSA; Lane 7: 40 μg Jurkat cell lysate; Lane 8: 5 μg GST fusion protein; Lane 9: Novex Sharp Unstained Protein Standard; Lane 10: 5 μg β-galactosidase.

stitutes tricine for glycine in the running buffer. The smaller proteins and peptides that migrate with the stacked DS ions

Recommended products

in the Tris-glycine gel system are well separated from DS ions

Use Novex Tricine gels with our In-Gel Tryptic Digestion Kit for separation and digestion of peptides for mass spectrometry.

in the Novex Tricine gel system, offering sharper bands and higher resolution.

 Learn more at thermofisher.com/tricine For ordering information refer to page 85. For quick reference on the protocol please refer to page 79.



Protein standards



Protein gel stains

Select precast gel


Select the standard


Run the gel

Stain the gel


Post stain

Novex IEF gels

21

Novex IEF gel

Precast gels for isoelectric point determination

Separated on precast vertical gel (slab) Cathode –

Isoelectric focusing (IEF) is an electrophoresis technique that separates proteins based on their isoelectric point (pI). The pI is the pH at which a protein has no net charge and does not move in an electric field. Invitrogen™ Novex™ IEF gels effectively create a pH gradient so proteins separate according to their unique pI (Figure 16 and 17). These gels can be used for pI determination or for detection of minor changes in a protein due to deamination, phosphorylation, or glycosylation, and can resolve different proteins of similar size that cannot be resolved on standard SDS-PAGE gels.

• Shelf life: 2 months

Did you know?

• Average run time: 2.5 hours

Harry Svensson-Rilbe and his student Olof Vesterberg first described the theory of separation of amphoteric proteins along a pH gradient by applying an electric field in the 1960s.

• Separation range: —pH 3–10 gels: pI performance range is 3.5–8 —pH 3–7 gels: pI performance range is 3.0–7.0 • Polyacrylamide concentration: fixed 5% • Gel dimensions: 8 x 8 cm (1 mm thick) • Maximum sample volume per 10-well gel: 20 μL

pl

1

2

3

4

5

6

7

8

9

• Higher resolution of slight differences in size when used in combination with SDS-PAGE for 2D electrophoresis

Anode +

10

8.0

• Accurate pI determination • Clear, sharp bands for easy identification of protein modifications




When used with our convenient, pre-optimized buffers, solubilizers, and molecular weight markers, Novex IEF gels can provide:

Specifications



20

7.4



th e

Mini Gel Ta

 nk

Figure 16. Novex IEF gel electrophoresis. A 2-fold dilution series 6.0 of IEF Marker 3–10 was run in duplicate on an Invitrogen™ 4.5 Novex™ pH 3–10 IEF Protein Gel using a Mini Gel Tank. The IEF Marker 3–10 consists of proteins with a variety of isoelectric points; these proteins include lectin (pI = 7.8, 8.0, and 8.3), myoglobin from horse muscle (pI = 6.9 and 7.4), carbonic anhydrase from bovine erythrocytes (pI = 6.0), β-lactoglobulin from bovine milk (pI = 5.2 and 5.3), soybean trypsin inhibitor (pI = 4.5), and glucose oxidase (pI = 4.2). After electrophoresis, the gel was fixed and stained using Coomassie R-250 dye. Gel imaging was performed with a flatbed scanner. Volume of IEF Marker 3–10 loaded: Lanes 1 and 6: 20 μL; Lanes 2 and 7: 10 μL; Lanes 3 and 8: 5 μL; Lanes 4 and 9: 2.5 μL; Lanes 5 and 10: blank.

Figure 17. Novex IEF gel migration chart using the Novex IEF marker. Proteins shown are 1: amyloglucosidase (Aspergillus niger), pI = 3.5; 2: glucose oxidase (Aspergillus niger), pI = 4.2; 3: trypsin inhibitor (soybean), pI = 4.5; 4a and 4c: β-lactoglobulin (bovine, milk), pI = 5.2 and 5.3; 5: carbonic anhydrase (bovine, erythrocytes), pI = 6.0; 6a and 6c: myoglobin (horse, muscle), pI = 6.9 and 7.4; 7a, 7m and 7c: lectin (Lens culinaris), pI = 7.8, 8.0 and 8.3; 8: ribonuclease A (bovine, pancreas), pI = 9.5; and 9: cytochrome c (horse, heart), pI = 10.7.

Recommended products Novex IEF buffer kits—includes optimized cathode, anode, and sample buffers to reduce variability and enable consistent results. IEF Marker 3–10—ready to use, enables accurate results.

ZOOM™ IEF Fractionator Combo Kit— offers a fast, reliable method to reduce sample complexity, enrich low-abundance proteins, and increase the dynamic range of detection.

 Learn more at thermofisher.com/ief For ordering information refer to page 85. For quick reference on the protocol please refer to page 79.



Protein standards



Protein gel stains

Select precast gel



Select the standard

Novex Zymogram gels

Run the gel

Stain the gel


Post stain

23

Table 1. Novex Zymogram gels available. Novex Zymogram gel Novex Zymogram gelatin gel

Easy in-gel protease analysis Invitrogen™ Novex™ Zymogram gels are excellent tools for detecting and characterizing proteases that utilize casein or gelatin as a substrate. Casein and gelatin are the most commonly used substrates for demonstrating the activity of proteases. Novex Zymogram gels are used to analyze a variety of enzymes, including matrix metalloproteinases, lipases, and other proteases (Figure 18). Available gel types are shown in Table 1.

Novex Zymogram casein gel

Novex Zymogram blue casein gel

Gel composition

10% TrisGlycine gel

12% TrisGlycine gel

4–16% TrisGlycine gel

Substrate

0.1% gelatin

0.05% casein

0.1% casein, with blue stain incorporated in gel

Protease analysis 10% gel (w/gelatin)

12% gel (w/casein)

4—16% gel (w/prestained casein blue)

10

20

Sensitivity

10–6 units of collagenase

7 x 10 –4 units of trypsin

1.5 x 10 –3 units of trypsin

How do Novex Zymogram gels work?

Post-staining required?

Yes

Yes

No

Protease samples are denatured in SDS buffer under nonre-

Separation range

20–120 kDa

30

Good to know

1

40

30–150 kDa

10–220 kDa

Zymogram gel using Novex Tris-Glycine SDS Running Buffer. After electrophoresis, the enzyme is renatured by incubating the gel in Invitrogen™ Novex™ Zymogram™ Renaturing Buffer

Specifications

that contains a nonionic detergent. The gels are then equili-


brated in Invitrogen™ Novex™ Zymogram™ Developing Buffer to add divalent metal cations required for enzymatic activity, and then stained and destained. Regions of protease activity appear as clear bands against a dark blue background where the protease has digested the substrate.

% of length of gel

ducing conditions and without heating, and run on a Novex

50 2 1

60

2

2

3 3

• Average run time: 90 minutes • Separation range: 10–220 kDa (Figure 19) • Polyacrylamide concentrations: fixed 10% (with gelatin), fixed 12% (with casein); gradient 4–16% (with blue casein)

70

Figure 19. Novex Zymogram gel migration chart. The numbered bands refer to the following proteases: Band 1: Collagenase Type I (140 kDa) Band 2: Thermolysin (37 kDa) Band 3: Chymotrypsin (30 kDa) Band 4: Trypsin (19 kDa)

4

3

80

4

2

• Gel dimensions: 8 x 8 cm (1 mm thick) • Maximum sample volume per well: 20 μL

2

3

4

5

6

7

8

9 10

4

90




1



22

Figure 18. Novex Zymogram gel electrophoresis. Type I collagenase was run in duplicate on an Invitrogen™ Novex™ 10% Zymogram (Gelatin) Protein   th e k Gel using a Mini Gel Tank. The gel was developed Mini Gel Tan using Novex Zymogram Renaturing Buffer and Novex Zymogram Developing Buffer and stained using SimplyBlue SafeStain. Images were acquired using a flatbed scanner. Lanes 3 and 7: 5 μL of 2.0 μU/mL type I collagenase; Lanes 1, 4, 5, and 10: 12 μL SeeBlue Prestained Protein Standard.

4

100

Recommended products After electrophoresis, incubate the gel in Zymogram Renaturing Buffer to renature the enzyme. The gels are then equilibrated in Zymogram Developing Buffer to add divalent metal cations required for enzymatic activity.

 Learn more at thermofisher.com/zymogram For ordering information refer to page 85. For quick reference on the protocol please refer to page 80.



Protein standards



Protein gel stains

24


Select precast gel

Select the standard


Run the gel

Stain the gel


Post stain

E-PAGE High-Throughput Precast Gel System

E-PAGE gel

E-PAGE 48 8% Gel

Protein separation and analysis for increased sample throughput

E-PAGE 96 6% Gel

0%

0% 220 kDa

10%

The Invitrogen E-PAGE High-Throughput Precast Gel System is designed for fast, bufferless medium- and high-throughput protein analysis. Invitrogen™ E-PAGE™ 48-well and 96-well precast gels consist of a buffered gel matrix and electrodes packaged inside a disposable, UV-transparent cassette. Each cassette is labeled with a unique barcode to facilitate identification of the gel using commercial barcode readers. These gels can be loaded by multichannel pipettor or automated loading system. The E-PAGE system also includes E-Base™ integrated devices to run the gels, an E-Holder™ platform for optional robotic loading, and free E-Editor™ 2.0 Software to align images for easy comparison. ™

220 kDa

™

Advantages of using the E-PAGE High-Throughput Precast Gel System include: • Ease-of-use—quick setup and fast protein separation in about 23 minutes

25

120 kDa

25%

Did you know?

20% 120 kDa 30%

100 kDa

60 kDa 40 kDa

80 kDa

75% 20 kDa

40%

Specifications

50%

60 kDa


50%

• Average run time: 14 minutes

50 kDa

100%

Figure 21. E-PAGE gel migration chart. Migration patterns of the Invitrogen™ E-PAGE™ MagicMark™ Unstained Protein Standard are shown.

Our E-Base devices are compatible with the Society for Biomolecules Screening (SBS) standard plate size and can be conveniently mounted on liquid handling robot decks.

40 kDa 60%


30 kDa

• Polyacrylamide concentrations: –– E-PAGE™ 48 gel: fixed 8%

70% 20 kDa

–– E-PAGE™ 96 gel: fixed 6%

80%

• Gel dimensions: 13.5 x 10.8 cm (3.7 mm thick) • Maximum sample volume per well: –– E-PAGE 48 gel: 20 µL –– E-PAGE 96 gel: 15 µL

90%

100%

B

A

• Fast loading—compatible with multichannel pipettors and robotic loading


• Efficient western blotting and staining—optimized protocols and reagents

The E-PAGE™ SeeBlue™ Prestained Protein Standard or E-PAGE MagicMark Unstained Protein Standard are specifically designed for use with E-PAGE gels.

Good to know

C Mother E-Base

E-PAGE 96 gels

How do E-PAGE gels work? E-PAGE gels run in the Invitrogen™ E-Base electrophoresis de-

Daughter E-Base

vice, which has an integrated power supply for direct connection to an electrical outlet. Use the Invitrogen™ Mother E-Base™ device for a single E-PAGE gel, or use the Mother E-Base device in conjunction with two or more Invitrogen™ Daughter

Figure 20. Loading and running E-PAGE gels. (A) Loading E-PAGE 48 gels using a multi-channel pipettor. (B) Loading E-PAGE 96 gels using a multi-channel pipettor. (C) The Mother/Daughter E-Base combination.

E-Base™ devices for running multiple gels simultaneously.

 Learn more at thermofisher.com/epage For ordering information refer to page 85.



Protein standards



Protein gel stains

Select precast gel


Select the standard


Run the gel

Stain the gel

Prepare the sample Sample prep kits

High salt concentration in samples: High salt concentrations result in increased conductivity that affects protein migration, and

Before a sample can be loaded onto a gel for analysis, it must be properly prepared. Depending on the gel type, this may involve denaturing the proteins, reducing any disulfide bonds, adjusting the ionic strength, and removing interfering contaminants. General guidelines for preparing samples are provided below. General guidelines for preparing samples: Prepare your sample in the appropriate sample buffer such that the final concentration of the sample buffer is 1X. Recommended sample buffers are listed on page 29. Running reduced and non-reduced samples: For optimal


Post stain

can result in gel artifacts in adjacent lanes containing samples with normal salt concentrations. Perform dialysis or precipitate and resuspend samples in lower-salt buffer prior to electrophoresis.

Pierce SDS-PAGE Sample Prep Kit

Sample treated with the Pierce SDS-PAGE Sample Prep Kit

Untreated M

S

M

S

M

S

M

Guanidine-HCl in samples: Samples solubilized in guanidineHCl have high ionic strength, and produce increased conductivity similar to high salt concentrations. In addition, guanidine precipitates in the presence of SDS leading to various types of gel artifacts. If possible, change the solubilization agent by dialysis prior to electrophoresis.

Cell lysates Consider the following when performing electrophoresis of cell lysates: • Genomic DNA in the cell lysate may cause the sample to become viscous and affect protein migration patterns and resolution. Shear genomic DNA to reduce viscosity before loading the sample. • Cells lysates contain soluble and insoluble fractions. The size of each fraction depends on the type of sample being analyzed. The nature of the insoluble fraction may result in altered protein migration patterns and resolution. Separate the two fractions by centrifugation and load them on separate lanes for electrophoresis. • If radioimmunoprecipitation assay (RIPA) buffer is used in cell lysis, subsequent blotting of proteins less than 40 kDa may be inhibited due to the presence of Triton™ X-100 in the buffer.

A protein sample can be purged of any contaminants typically in only 10 minutes using the Thermo Scientific™ Pierce™ SDS-PAGE Sample Prep Kit. This is much faster than dialysis or ultrafiltration and yields higher protein recoveries while concentrating the sample. Advantages of using the Pierce SDS-PAGE Sample Prep Kit include: • Eliminates artifacts caused by incompatible contaminants—removes dyes, reducing agents, detergents, sugars, glycerol, guanidine, urea, and ammonium sulfate to provide reproducible results for SDS-PAGE analysis (Figure 22) • Compatible with the Thermo Scientific™ Pierce™ BCA Assay—allows quantification of the processed sample • Enriches dilute protein solutions—concentrates protein sample by eight-fold in less than 20 minutes for SDS-PAGE analysis (Figure 22) • Fast and easy to use for up to 70 μg of protein per sample—uses new spin cup format that allows higher amounts of protein to be processed than with the original procedure

Figure 22. Minimize distortion caused by detergents. Rat C6 cells were lysed and a membrane protein fraction isolated using the Thermo Scientific™ MemPER™ Eukaryotic Membrane Protein Extraction Reagent. Membrane and hydrophilic cell fractions were separated by SDS-PAGE using 4–20% gradient gels with or without prior treatment using the Pierce SDS-PAGE Sample Prep Kit. Western blot analysis was performed using an antibody against cytochrome oxidase subunit 4 (COX4) and Thermo Scientific™ SuperSignal™ West Femto chemiluminescent substrate. Kit-treated samples exhibit better band straightness and resolution with low molecular weight proteins than samples that were untreated. S = Soluble fraction (hydrophilic) M = Membrane fraction 100

Figure 23. Consistent 88% 80 protein 85% 77% 77% recovery is 75% 74% achieved 60 using the Pierce SDS40 PAGE Sample Prep Kit. 20 Pure proteins (60 µg) of 0 assorted Carbonic Ovalbumin Transferrin Ubiquitin Cytochrome c Bacterial anhydrase lysat e molecular mass (30, 44, 80, 86, and 120 kDa) as well as a bacterial lysate were processed using this kit. Protein concentrations were determined with the Pierce BCA Protein Assay Kit and reported as percent protein recovered. Table 2. Interfering substances effectively removed.

Good does to know How it work?

For quick protein clean-up and enrichment for SDS-PAGE we

samples on the same gel. If you do choose to run reduced and

recommend using the Thermo Scientific Pierce SDS-PAGE Sample

non-reduced samples on the same gel, do not run reduced and

Prep Kit, which removes substances such as guanidine-HCL

Our Pierce SDS-PAGE Sample Prep Kit uses a unique resin

non-reduced samples in adjacent lanes. The reducing agent may

and ionic detergents that can result in protein bands that appear

of modified diatomaceous earth that binds protein in DMSO.

have a carry-over effect on the non-reduced samples if they are in

smeared or wavy in the gel or on a western blot.

Simply combine 2–300 μL of sample containing up to 70 μg of

Interfering reagents

Percent protein recovered (Starting amount = 20 µg BSA)

Control (water)

75%

0.5 M Sodium chloride

80%

2 M Ammonium sulfate

76%

protein with 20 μL of Pierce™ SDS Protein Binding Resin and

20% SDS

75%

DMSO. After the proteins bind to the resin, wash away the

10% Triton™ detergent

75%

unbound contaminating chemicals. Finally, elute the sample

6 M Urea:DMSO (1:3 ratio)

75%

buffer results in proteolysis (Kubo, 1995). We recommend heating

in 50 μL of the Elution Buffer. The recovered protein sample is

1 M Sodium chloride

75%

samples for denaturing electrophoresis (reduced or non-reduced)

ready to mix with the supplied Sample Loading Buffer for

6 M Urea

74%

at 85°C for 2–5 minutes for optimal results. Do not heat the

gel loading.

10% CHAPS

80%

25% Glycerol

71%

10% OTG

71%

2 M Guanidinium•HCl

70%

40% Sucrose

70%

Heating samples: Heating the sample at 100°C in SDS-containing

samples for non-denaturing (native) electrophoresis or Novex Zymogram Gels.

 Learn more at thermofisher.com/PAGEsampleprep

For ordering information refer to page 85.

S

Quick protein clean-up and enrichment for SDS-PAGE

results, we do not recommend running reduced and non-reduced

close proximity.

27

Percent protein recovered

26



Protein standards



Protein gel stains

28

Select precast gel


Select the standard


Run the gel

Stain the gel

Select buffers

29

Recommended SDS-PAGE buffers and reagents

Buffers and reagents

Protein samples prepared for PAGE analysis are denatured by heating in the presence of a sample buffer with or without a reducing agent. The protein sample is mixed with the sample buffer and heated for 2–10 minutes, then cooled to room temperature before it is applied to the sample well on the gel. Loading buffers also contain glycerol so that they are heavier than water and sink neatly to the bottom of the buffer-submerged well when added to a gel.


Post stain

Gel type

If suitable, negatively charged, low molecular weight dye is also included in the sample buffer; it will migrate at the buffer front, enabling one to monitor the progress of electrophoresis. The most common tracking dye for sample loading buffers is bromophenol blue. We offer premixed, reliable SDS-PAGE buffers and reagents including sample buffers, running buffers, reducing agents, and antioxidants.

 Learn more at

Bolt Bis-Tris Plus Gel

Sample buffer optimized for use with the gel

Other compatible sample buffers

Running buffer optimized for use with the gel

• Bolt™ Sample Reducing Agent (10X) • Bolt™ LDS Sample Buffer (4X) (nonreducing) • Bolt Antioxidant

• Pierce™ LDS Sample Buffer (4X) for storage at RT

• Bolt™ MES SDS Running Buffer (20X) • Bolt™ MOPS SDS Running Buffer (20X)

NuPAGE Bis-Tris Gel

• NuPAGE Sample Reducing Agent (10X) • NuPAGE Antioxidant • NuPAGE™ LDS Sample Buffer (4X) (nonreducing)

NuPAGE Tris-Acetate Gel

• Novex Tris-Glycine SDS Sample Buffer (2X) • NuPAGE Sample Reducing Agent (10X) • Novex Tris-Glycine Native Sample Buffer (2X)

Novex Tris-Glycine Gel

• Novex Tris-Glycine SDS Sample Buffer (2X) • NuPAGE Sample Reducing Agent • Novex Tris-Glycine Native Sample Buffer (2X)

™

• Pierce™ Lane Marker Non• NuPAGE™ MES SDS Reducing Sample Running Buffer (20X) Buffer (5X)— • NuPAGE™ MOPS SDS storage at RT; Running Buffer (20X) when you desire to dilute your sample less and require transferable marker dye to nitrocellulose membranes • Pierce™ Lane Marker Reducing Sample Buffer (5X)—when you desire to dilute your sample less and require transferable marker dye to nitrocellulose membranes

MES vs. MOPS Running Buffer: • Use MES SDS running buffers to resolve small molecular weight proteins. • Use MOPS running buffers to resolve mid-size proteins. MES has a lower pKa than MOPS, enabling gels with MES running buffer to run faster than gels with MOPS SDS running buffer. The difference in ion migration affects stacking and results in a difference in protein separation range between these buffers.

• NuPAGE™ Tris-Acetate SDS Running Buffer (20X) • Novex Tris-Glycine Native Running Buffer (10X)

Reducing agent: When preparing samples for reducing gel electrophoresis, any of the following reducing agents may be used: •B olt Sample Reducing Agent

• Novex Tris-Glycine SDS Running Buffer (10X) • Novex Tris-Glycine Native Running Buffer (10X) • Pierce™ Tris-Glycine SDS Buffer (10X) • BupH™ Tris-Glycine-SDS Buffer Packs

•N uPAGE Sample Reducing Agent

Novex Tricine Gel

• Novex™ Tricine SDS Sample Buffer (2X)

• Novex Tricine SDS Running Buffer (10X)

NativePAGE Gel

• NativePAGE™ Sample Buffer (4X) • NativePAGE™ 5% G-250 Sample Additive

• NativePAGE™ Running Buffer (20X) • NativePAGE™ Cathode Buffer Additive (20X)

Novex IEF Gel

• Novex™ IEF Sample Buffer, pH 3–10 (2X) • Novex™ IEF Sample Buffer, pH 3–7 (2X)

• Novex™ IEF Anode Buffer (50X) • Novex™ IEF Cathode Buffer, pH 3–10 (10X) • Novex™ IEF Cathode Buffer, pH 3–7 (10X)

Novex Zymogram Gels*

• Novex Tris-Glycine SDS Sample Buffer (2X)

• Novex Tris-Glycine SDS Running Buffer (10X)

ithiothreitol (DTT), 50 mM final D concentration • β-mercaptoethanol (β-ME), 2.5% final concentration • tris(2-carboxyethyl)phosphine (TCEP), 50 mM final concentration Add the reducing agent to the sample up to an hour before loading the gel. Avoid storing reduced samples for long periods, even if they are frozen. Reoxidation of samples can occur during storage and produce inconsistent results.

*Novex Zymogram Developing Buffer (10X) and Novex Zymogram Renaturing Buffer (10X) are available for visualizing the Novex Zymogram gels.

thermofisher.com/electrophoresisbuffers For ordering information refer to page 85.



Protein standards



Protein gel stains

30

Select precast gel


Select the standard


Run the gel

Stain the gel


Post stain

31

Buffer recipes NuPAGE buffer recipes

Tris-glycine buffer recipes

Buffer

Storage

NuPAGE LDS Sample Buffer

+4˚–25˚C

NuPAGE MOPS SDS Running Buffer*

Component

Concentration (1X)

Glycerol Tris base Tris HCl LDS EDTA SERVA™ Blue G-250 Phenol Red

0% 141 mM 106 mM 2% 0.51 mM 0.22 mM 0.175 mM (pH 8.5)

NuPAGE Tris-Acetate SDS Running Buffer

Tris-Glycine SDS Sample Buffer

+4˚C

Tris-Glycine Native Sample Buffer

50 mM 50 mM 0.1% 1 mM (pH 7.7)

Tris-Glycine SDS Running Buffer

50 mM 50 mM 0.1% 1 mM (pH 7.3)

Tris-Glycine Native Running Buffer

Room temperature

Room temperature

+4˚–25˚C Bicine Bis-Tris (free base) EDTA Chlorobutanol

25 mM 25 mM 1.0 mM 0.05 mM (pH 7.2)

Tris base Tricine SDS

50 mM 50 mM 0.1% (pH 8.24)

Tris-Glycine Transfer Buffer

Component

Concentration (1X)

Tris HCl* Glycerol SDS Bromophenol Blue Deionized water

63 mM 10% 2% 0.0025% — (pH 6.8)

Tris HCl* Glycerol Bromophenol Blue Deionized water

100 mM 10% 0.0025% — (pH 8.6)

Tris base Glycine SDS Deionized water

25 mM 192 mM 0.1% — (pH 8.3)

Tris base Glycine Deionized water

25 mM 192 mM — (pH 8.3)

Tris base Glycine Deionized water

12 mM 96 mM — (pH 8.3)

+4˚C

+4˚–25˚C MES Tris base SDS EDTA

NuPAGE™ Transfer Buffer

Storage

+4˚–25˚C MOPS Tris base SDS EDTA

NuPAGE MES SDS Running Buffer*

Buffer

Room temperature

+4˚–25˚C * Tris HCl solutions are prepared from Tris base and pH adjusted with 6 N HCl.

* The pre-mixed buffers (Cat. Nos. NP0001 and NP0002) also contain trace amounts of the proprietary NuPAGE Antioxidant (Cat. No. NP0005) for stability. Additional Antioxidant may be required with specific protocols.




Protein standards



Protein gel stains

32

Select precast gel


Select the standard


Run the gel

Stain the gel


Post stain

33

Buffer recipes Isoelectric focusing buffer recipes

Tricine buffer recipes Buffer

Storage

Tricine SDS Sample Buffer

+4˚C

Tricine SDS Running Buffer

Room temperature

Component

Concentration (1X)

Tris HCl* Glycerol SDS Coomassie Blue G Phenol Red Deionized water

450 mM 12% 4% 0.0075% 0.0025% – (pH 8.45)

Tris base Tricine SDS Deionized water

100 mM 100 mM 0.1% – (pH 8.3)

Buffer

Storage

IEF Sample Buffer pH 3-7

+4˚C

IEF Sample Buffer pH 3-10

+4˚C

* Tris HCl solutions are prepared from Tris base and pH adjusted with 6 N HCl.

IEF Cathode Buffer pH 3-10 (upper buffer chamber)

+4˚C

Zymogram buffer recipes

IEF Anode Buffer (for both pH ranges) (lower buffer chamber)

Room temperature

Urea-Thiourea-CHAPS (rehydration buffer for IPG strips)

–20˚C

Storage

Zymogram Renaturing Buffer

Room temperature

Zymogram Developing Buffer

Room temperature

* Tris HCl solutions are prepared from Tris base and pH adjusted with 6 N HCl.


Component

Concentration (1X)

Triton™ X-100 Deionized water

2.7% (w/v) in H2O

Tris HCI* NaCl CaCl2•2 H2O Brij™ 35 Deionized water

50 mM 200 mM 5 mM 0.006% (w/v) _ (pH 7.6)

Concentration (1X)

Lysine (free base)

40 mM

Glycerol Deionized water

15% —

Arginine (free base) Lysine (free base) Glycerol Deionized water

20 mM 20 mM 15% —

Lysine (free base) Deionized water

40 mM —

Arginine (free base) Lysine (free base) Deionized water

20 mM 20 mM — (pH 10.1)

Phosphoric acid 85% Deionized water

7 mM — (pH 2.4)

Deionized urea Deionized thiourea CHAPS Ampholytes* Bromophenol Blue

7M 2M 2–4% 0.2–2.0% 0.002%

Ultrapure water

—

DTT

20 mM

+4˚C

IEF Cathode Buffer pH 3-7 (upper buffer chamber)

Buffer

Component

* For ZOOM™ Strip pH 9-12 use 1% ZOOM™ Focusing Buffer pH 7-12 instead of ampholytes.



Protein standards



Protein gel stains

34

Select precast gel


Select the standard


Run the gel

Stain the gel


Post stain

35

Select the standard Protein ladders and standards To assess the relative molecular weights (sizes) of proteins in a sample, a mixture containing several proteins of known molecular mass are run alongside the test sample lane(s). Often these protein mixtures are run on the outer lanes of the gel, to maximize the number of remaining gel wells for test samples, but can also be useful in the middle wells of the gel when running a large gel with many wells. Such sets of known protein mixtures are called protein molecular weight markers or protein ladders. It is important to choose a protein ladder that consists of proteins with molecular weights that span the molecular weight range of the protein(s) of interest. A standard curve can be constructed from the distances

each marker protein migrates through the gel. After measuring the migration distance that an unknown protein travels through the same gel, its molecular weight can be determined graphically from the standard curve. Several kinds of ready-to-use protein molecular weight (MW) markers are available that are labeled, prestained, or unstained for different modes of detection and downstream applications. We offer ladders suitable for both SDSPAGE as well as native PAGE.

Unstained protein ladders

Ready-to-use prestained and unstained protein ladders with exceptional lot-to-lot consistency

Low range

PageRuler Unstained Low Range Protein Ladder

Broad range

PageRuler Unstained Protein Ladder

We offer a broad range of prestained and unstained protein

High range

NativeMark Unstained Protein Standard

ladders supplied in a ready-to-use format to facilitate easy protein

Recommended for: • Precise determination of target protein molecular weight

analysis during gel electrophoresis and western blotting (Table 3). All of our protein ladders offer: • Performance—sharp protein bands and consistent migration patterns provide easy molecular weight determination

Prestained protein ladders Low range

PageRuler Prestained Protein Ladder

• Convenience—protein ladders are ready to load, with no heating required

Broad range

PageRuler Plus Prestained Protein Ladder Spectra™ Multicolor Broad Range Protein Ladder

• Reliability—exceptional lot-to-lot consistency and reproducibility

High range

HiMark Prestained Protein Standard Spectra Multicolor High Range Protein Ladder

Recommended for: • Approximate determination of molecular weight • Monitoring the progress of electrophoresis runs • Estimating the efficiency of protein transfer to the membrane during western blotting

 Learn more at thermofisher.com/proteinstandards

Other Western

MagicMark XP Western Protein Standard

Specialty

PageRuler Prestained NIR Protein Ladder BenchMark Fluorescent Protein Standard BenchMark His-tagged Protein Standard IEF Marker 3-10



Protein standards



Protein gel stains

36

Select precast gel


Select the standard


Run the gel

Stain the gel


Post stain

37

Table 3. Protein standard selection guide Category

Range

No. of bands

Reference bands

Protein MW determination

Protein band visualization

Monitoring electrophoresis run

Coomassie dye, silver, Monitoring protein or fluorescent staining transfer

Chemiluminescent band visualization


3.4–100 kDa

8

25 kDa

Best

NA

NA

Best

NA

Good


10–200 kDa

14

50 kDa

Good

NA

NA

Good

NA

Good


20–1,200 kDa

8

Best for native electrophoresis

NA

NA

Best

NA

Good


10–180 kDa

10

Green 10 kDa; orange 70 kDa

Good

Good

Good

NA

Good

Good

PageRuler Plus Prestained Protein Ladder

10–250 kDa

9

Green 10 kDa; orange 25 and 70 kDa

Good

Good

Good

NA

Good

NA

HiMark Prestained Protein Standard

30–460 kDa

9

Best for high MW proteins

Good

Good

NA

Best for high MW proteins

NA

Product

Unstained ladders and standards Unstained standards

Pretained protein ladders Prestained protein standards

Spectra Multicolor Broad Range 10–260 kDa Protein Ladder

10

Green 10 and 50 kDa; orange 40, 70, and 260 kDa; pink 140 kDa

Good

Best

Best

NA

Best

NA

Spectra Multicolor High Range Protein Ladder

40–300 kDa

8

Green 50 kDa; orange 70 and 300 kDa

Good

Best

Best

NA

Best

NA

Other ladders and standards IEF

IEF Marker 3-10

pI 3.5–10.7

13

Best for pI estimation

NA

NA

Good

NA

NA

Chemiluminescent standard

MagicMark XP Western Protein Standard

20–220 kDa

9

Good

NA

NA

Good

NA

Best

Near infrared (NIR) standard

PageRuler Prestained NIR Protein Ladder

11–250 kDa

10

Good

NA

NA

NA

NA

NA

Fluorescent standard

BenchMark Fluorescent Protein Standard

11–155 kDa

7

Good

NA

NA

NA

NA

Good

His-tag standard

BenchMark His-tagged Protein Standard

10–160 kDa

10

Best

NA

NA

Good

NA

Good for detection with anti-His antibody

55 kDa

 Learn more at thermofisher.com/proteinstandards




Protein standards



Protein gel stains

38

Select precast gel


Select the standard


Run the gel

Stain the gel


Post stain

39

Unstained ladders and standards



Sharp bands and precise molecular weight estimation for low molecular weight proteins

Sharp bands and precise molecular weight estimation for a wide range of proteins

Thermo Scientific™ PageRuler™ Unstained Low Range Protein Ladder is a mixture of eight proteins and peptides for use as size standards that resolve into clearly identifiable sharp bands when analyzed by SDS-PAGE. The proteins (except for the 5 and 3.4 kDa peptides) contain an integral Strep-tag™ II Sequence and may be detected on western blots using Strep-Tactin™ Conjugates. • Comprehensive—eight proteins and peptides spanning 3.4 to 100 kDa; the 25 kDa band is more intense than the other bands for easy orientation • Versatile—compatible with western blots by staining with Ponceau S dye or Coomassie dye; compatible with Thermo Scientific™ Pierce™ Reversible Protein Stain Kit for Nitrocellulose Membranes or other protein stains

PageRuler Unstained Low Range Protein Ladder NuPAGE 4–12% Bis-Tris Gel with MES SDS buffer

Storage specifications • Storage buffer: Tris-H3PO4, EDTA, SDS, DTT, sodium azide, bromophenol blue, and glycerol • Storage conditions: upon receipt store at –20°C • Stability: 1 year from date of receipt

Recommended products The PageRuler Unstained Protein Ladder is recommended for Novex Tris-Glycine, Bis-Tris or Tris-Acetate gels.

Thermo Scientific™ PageRuler™ Unstained Protein Ladder is a mixture of 14 recombinant, highly purified, unstained proteins for use as size standards in SDS-PAGE and western blotting. Each protein in the ladder contains an integral Strep-tag II Sequence, which can be detected directly on western blots using a Strep-Tactin Conjugate or an antibody against the Strep-tag II Sequence. • Comprehensive—14 highly purified proteins with excellent accuracy spanning 10 to 200 kDa; the ladder contains one 50 kDa reference band of higher intensity • Versatile—compatible with Coomassie dye; compatible with Pierce Reversible Protein Stain Kit for Nitrocellulose Membranes, silver staining, or western blotting

 Learn more at


Recommended products The PageRuler Unstained Protein Ladder is recommended for Novex Tris-Glycine, Bis-Tris or Tris-Acetate gels.

 Learn more at

thermofisher.com/unstainedstandards


PageRuler Unstained Protein Ladder NuPAGE 4–12% Bis-Tris Gel with MES SDS buffer




Protein standards



Protein gel stains

40


Select precast gel

Select the standard


Run the gel

Stain the gel


Post stain

41

Prestained ladders

NativeMark Unstained Protein Standard Convenient molecular weight estimation for native electrophoresis

kDa kDa

1,0481,048 1,2361,236 1,0481,048

720 720 480 480

720 720 242 242 480 480 146 146 242 242

The Invitrogen NativeMark Unstained Protein Standard is designed for molecular weight estimation of proteins using native gel electrophoresis. ™

™

• Comprehensive—contains a wide range of high molecular weight proteins, providing 8 protein bands in the range of 20–1,200 kDa • Versatile—can be visualized using Coomassie, silver, or fluorescent stains after electrophoresis, or with Ponceau S, Coomassie, or other membrane stains after western transfer


kDa kDa 1,2361,236

66 66

146 146

NativeMark Unstained Protein Standard NativePAGE Bis-Tris gels

66 66 20 20 20 20

3–12% 3–12%

4–16% 4–16%

Storage specifications • Storage buffer: Bis/Tris-HCl (pH 7.0), NaCl, glycerol, and Ponceau S • Storage conditions: upon receipt store at –20°C • Stability: 6 months

Recommended products The NativeMark Unstained Protein Standard is recommended for use with NativePAGE Bis-Tris gels, Novex Tris-Glycine gels, or NuPAGE Tris-Acetate gels.

Outstanding clarity for easy molecular weight determination of low molecular weight proteins Thermo Scientific™ PageRuler™ Prestained Protein Ladder is a mixture of 10 blue-, orange-, and green-stained proteins for use as size standards in SDS-PAGE and western blotting. The mobility of prestained proteins can vary in different SDSPAGE buffer systems; however, they are suitable for approximate molecular weight determination when calibrated against unstained standards in the same system. • Comprehensive—contains 10 proteins with a range of 10 to 180 kDa; includes one 70 kDa reference protein colored with an orange dye and one 10 kDa reference protein colored with a green dye

PageRuler Prestained Protein Ladder NuPAGE 4–12% Bis-Tris Gel with MES SDS buffer


Recommended products The PageRuler Prestained Protein Ladder is recommended for use with Tris-glycine, Bis-Tris, and Tris-acetate gels.

• Versatile—compatible with Coomassie dye staining and western blotting

 Learn more at

 Learn more at

thermofisher.com/prestainedstandards





Protein standards



Protein gel stains

42

Select precast gel


Select the standard


Run the gel

Stain the gel

PageRuler Plus Prestained Protein Ladder Outstanding clarity for easy molecular weight determination of a broad range of proteins


Post stain

HiMark Prestained Protein Standard PageRuler Plus Prestained Protein Ladder NuPAGE 4–12% Bis-Tris Gel with MES SDS buffer

• Comprehensive—9 proteins with a broad range of 10 to 250 kDa; includes 70 kDa and 25 kDa reference proteins that are colored with an orange dye and one 10 kDa reference protein that is colored with a green dye

kDa

198

220

98

268 238

62

171

117

• Storage buffer: Tris-H3PO4, EDTA, SDS, DTT, sodium azide, bromophenol blue, and glycerol • Storage conditions: upon receipt store at –20°C

110 80

60

50

60 40 30

40

28

20

71

17

15 HiMark Prestained Protein Standard NuPAGE 3–8%10Tris-acetate SDS buffer 30

14

Standard

160

80

50

38

™

260

120 100

49

The Invitrogen HiMark Prestained Protein Standard is designed for analysis of high molecular6 3 weight proteins on NuPAGE Tris-acetate gels.

Storage specifications

kDa

kDa

460

Superb analysis of high molecular weight proteins ™

Thermo Scientific™ PageRuler™ Plus Prestained Protein Ladder is a mixture of 9 blue-, orange-, and green-stained proteins for use as size standards in SDS-PAGE and western blotting. The mobility of prestained proteins can vary in different SDS-PAGE buffer systems; however, they are suitable for approximate molecular weight determination when calibrated against unstained standards in the same system.

kDa

43

55

20

41

3.5

31

SeeBlue® Plus2

HiMark™ Pre-stained

MagicMark™ XP

Novex® Sharp Pre-stained

• Comprehensive—contains a wide range of high molecular Cat. No. LC5800 LC5602 LC5925 LC5699 weight proteins, providing 9 protein bands in the range of NuPAGE 3–8% NuPAGE NuPAGE NuPAGE Bis-Tris Gel, Storage Tris-Acetatespecifications Gel blotted to 4–12% Bis-Tris 4–12% Bis-Tris w/ Tris-acetate nitrocellulose, Gel w/ MES Gel w/ MES 30–460 kDa SDS buffer detected w/ SDS buffer SDS buffer WesternBreeze • Storage buffer: Tris-HCl, formamide, SDS, and phenol red Chemiluminescent Kit • Versatile—easy visualization of band migration during • Storage conditions: upon receipt store at –20°C electrophoresis and rapid evaluation of western transfer efficiency • Stability: 6 months from date of receipt ®

®

®

®

®

• Stability: 1 year from date of receipt

Recommended products Recommended products

The HiMark Prestained Protein Standard is recommended for use with NuPAGE Tris-Acetate gels under denaturing conditions. This standard can also be used with NuPAGE 4–12% Bis-Tris gels with Invitrogen™ NuPAGE MOPS SDS Running Buffer and Novex 4% Tris-Glycine gels. However, to obtain the best results with high molecular weight proteins, always use NuPAGE Tris-Acetate gels.

The PageRuler Plus Prestained Protein Ladder is recommended for Trisglycine, Bis-Tris, and Tris-acetate gels.


 Learn more at

The HiMark Prestained Protein Standard is also available as part of the following kits that include gels, running and sample buffers, and stains or blotting materials: • Invitrogen™ NuPAGE™ Large Protein Staining Kit • Invitrogen™ NuPAGE™ Large Protein Sensitive Staining Kit • Invitrogen™ NuPAGE™ Large Protein Blotting Kit


 Learn more at thermofisher.com/prestainedstandards




Protein standards



Protein gel stains

44

Select precast gel


Select the standard


Run the gel

Stain the gel


Post stain

Spectra Multicolor Broad Range Protein Ladder


Superior visualization and analysis of a broad range of proteins

Superior and convenient visualization of high molecular weight proteins

Thermo Scientific Spectra Multicolor Broad Range Protein Ladder is a 4-color protein standard containing 10 prestained proteins for use in gel electrophoresis and western blotting. This standard is designed for monitoring the progress of gels during SDS-PAGE and for assessing western blot transfer efficiency. Four different chromophores (blue, orange, green, and pink) are bound to the different component proteins, producing a brightly colored ladder with an easy-to- pattern. ™

Spectra Multicolor Broad Range Protein Ladder NuPAGE 4–12% Bis-Tris Gel with MES SDS buffer

™

• Comprehensive—10 proteins with similar intensity spanning a broad range of 10 to 260 kDa • Versatile—compatible with Coomassie dye staining and western blotting

Thermo Scientific Spectra Multicolor High Range Protein Ladder is a mixture of 8 blue-, green-, and orange-stained proteins for use as size standards for high molecular weight proteins in gel electrophoresis and western blotting. This marker is designed for monitoring the progress of gels during SDS-PAGE, assessing western blot transfer efficiency, and estimating the approximate size of proteins after gel staining or western blotting. ™


Recommended products The Spectra Multicolor Broad Range Protein Ladder is recommended for Tris-glycine, Bis-Tris, and Tris-acetate gels.

™

• Comprehensive—8 proteins of similar intensity spanning a range of 40 to 300 kDa; 3 different chromophores (blue, orange, and green) are bound to the different component proteins, producing a brightly colored ladder with an easy-to- pattern

Spectra Multicolor High Range Protein Ladder NuPAGE 4–12% Bis-Tris Gel with MES SDS buffer


Recommended products The Spectra Multicolor High Range Protein Ladder is recommended for Tris-glycine, Bis-Tris, and Tris-acetate gels.


 Learn more at

 Learn more at



45




Protein standards



Protein gel stains

46

Select precast gel



Select the standard

Run the gel

Stain the gel


Post stain

47

Other ladders and standards

MagicMark XP Western Protein Standard kDa

kDa


kDa

kDa

460

198

220

98

268 238

62

171

Accurate molecular weight estimation directly on western blots

260 160

120 100

49

117

80

110 80

60

50

60 40

50

38

30

40

28

MagicMark XP Western Protein Standard 20 NuPAGE Bis-Tris gel, blotted to nitrocellulose, 15 and detected with Invitrogen™ WesternBreeze™ 10 Chemiluminescent Kit

71

17

30

14

55

The MagicMark™ XP Western Protein Standard 20 6 3.5 is specifically designed for easy and convenient 41 3 protein molecular weight estimation directly on 31 HiMark SeeBlue MagicMark XP Novex Sharp Standard Pre-stained western blots. Each recombinant protein Plus2 in the Pre-stained Cat. No. LC5800 LC5602 LC5925 LC5699 Storage specifications standard contains an IgG binding site, which NuPAGE 3–8% NuPAGE NuPAGE NuPAGE Bis-Tris Gel, Tris-Acetate Gel blotted to 4–12% Bis-Tris 4–12% Bis-Tris binds the primary or secondary antibody used w/ Tris-acetate • nitrocellulose, Gel w/ MES Gel w/ MES S torage buffer: Tris-HCl (pH 6.8), DTT, glycerol, SDS, and detected w/ SDS buffer SDS buffer WesternBreeze for detection of the target protein, allowing direct SDS buffer Chemiluminescent bromophenol blue Kit visualization of the standard on the western blot. ™

®

®

®

®

™

®

®

®

• Storage conditions: upon receipt store at –20°C

• Comprehensive—consists of 9 recombinant proteins from 20 to 220 kDa • Versatile—compatible with chemiluminescent, chromogenic, and fluorescent detection

• Stability: 4 months from date of receipt

Recommended products The MagicMark XP Western Protein Standard is compatible with a broad range of gels—NuPAGE Bis-Tris gels, Novex Tris-Glycine gels, Novex Tricine gels, NuPAGE Tris-Acetate gels, and Bolt Bis-Tris Plus gels.

Sharp prestained standard for near-IR fluorescent visualization and protein sizing Thermo Scientific™ PageRuler™ Prestained NIR Protein Ladder is a mixture of 10 proteins that are stained blue and labeled with a fluorophore for near-infrared (NIR) fluorescent visualization and protein sizing following electrophoresis. The molecular weight markers in this ladder resolve into sharp bands when analyzed by SDS-PAGE. The 55 kDa band is of greater intensity and serves as a reference band. • Comprehensive—10 protein bands spanning 11 to 250 kDa • Versatile—visualize using instruments equipped for detection of near-infrared fluorescence such as certain Typhoon™ Imagers and the LI-COR Odyssey™ Infrared Imaging System; bands are directly visible because the proteins are prestained blue

 Learn more at

Visual detection

Infrared detection


Recommended products The PageRuler Prestained NIR Protein Ladder is recommended for visual detection, infrared imaging detection, and western blotting.

 Learn more at

thermofisher.com/westernblotstandard


PageRuler Prestained NIR Protein Ladder NuPAGE 4–12% Bis-Tris Gel with MES SDS buffer

thermofisher.com/specialtystandards



Protein standards



Protein gel stains

48


Select precast gel

Select the standard


Run the gel

Stain the gel


Post stain

BenchMark Fluorescent Protein Standard


Efficient estimation of molecular weight by fluorescent detection

Convenient detection and protein sizing of His-tagged proteins

BenchMark Fluorescent Protein Standard NuPAGE 4–12% Bis-Tris Gel with MES SDS buffer

The Invitrogen BenchMark Fluorescent Protein Standard consists of Alexa Fluor™ 488 dye– conjugated proteins for molecular weight estimation of fluorescently labeled proteins. ™

Storage specifications

The Invitrogen BenchMark His-tagged Protein Standard can be used as a positive control and for molecular weight sizing in His-tagged fusion protein detection. Each protein in the standard has a 6xHis tag.

• Storage buffer: Tris-HCl, SDS, glycerol, and Coomassie Blue G-250

• Comprehensive—10 sharp and clear bands from 10 to 160 kDa for molecular weight estimation of His-tagged proteins

™

• Comprehensive—consists of 7 distinct protein bands in the range of ~11–155 kDa • Versatile—visualize on a UV transilluminator or laser-based scanning instrument after SDS-PAGE

™

• Storage conditions: upon receipt store at –20°C • Stability: 6 months from date of receipt

Recommended products The BenchMark Fluorescent Protein Standard is recommended for use with NuPAGE gels or Novex Tris-Glycine gels.

BenchMark His-tagged Protein Standard NuPAGE 4–12% Bis-Tris Gel with MES SDS buffer

™

• Versatile—can be visualized with Invitrogen™ InVision™ HisTag In-Gel Stain or Coomassie R-250 stain on SDS-PAGE gels, or with Anti-His (C-term) Antibody using chromogenic or chemiluminescent detection systems

49

SimplyBlue stain

InVision stain

Storage specifications • Storage buffer: Tris-HCl, SDS, glycerol, DTT, and Coomassie Blue G-250 • Storage conditions: upon receipt store at –20°C • Stability: 6 months from date of receipt

Recommended products The BenchMark His-tagged Protein Standard is recommended for use with NuPAGE gels and Novex Tris-Glycine gels.

 Learn more at thermofisher.com/specialtystandards

 Learn more at thermofisher.com/specialtystandards

IEF Marker 3-10 Accurate determination of protein isoelectric points The IEF Marker 3-10 is a ready-to-use protein standard developed for IEF applications. This marker can be used for monitoring of protein separation on IEF gels and pI determination of unknown protein samples. • Comprehensive—13 purified isoforms from pI 3.5–10.7; no additional high range or low range markers are required • Versatile—can be used for both native and denaturing conditions

IEF Marker 3-10 Novex pH 3–10 Gel

Storage specifications • Storage buffer: 10% glycerol containing bromophenol blue (0.01%) and methyl red (0.01%) • Storage conditions: upon receipt store at –20°C • Stability: 1 year from date of receipt

 Learn more at


thermofisher.com/iefstandards

The IEF Marker 3-10 is applicable to all IEF gels (vertical or horizontal).




Protein standards



Protein gel stains

50

Select precast gel


Select the standard


Run the gel

Stain the gel


Post stain

51

Electrophoresis chambers and power supplies Electrophoresis run considerations: In electrical , the process of electrophoresis is closely associated with the following equations derived from Ohm’s Law: Voltage = Current × Resistance (V = IR) Wattage = Current × Voltage (W = IV)

Which electrophoresis chamber system is right for you?

Resistance

Current

The electrical resistance of the assembled electrophoresis cell is dependent on buffer conductivity, gel thickness, temperature, and the number of gels being run. Although the resistance is determined by the gel system, the resistance varies over the course of the run.

For a given gel/buffer system, at a given temperature, current varies in proportion to the field strength (voltage) and crosssectional area (thickness and number of gels). When using a constant current setting, migration starts slow, and accelerates over time, thus favoring stacking in discontinuous gels.

• In discontinuous buffer systems (and to a lesser extent in continuous buffer systems) resistance increases over the course of electrophoresis. This occurs in the Tris-glycine buffer system as highly conductive chloride ions in the gel are replaced by less conductive glycine ions from the running buffer.

When running under constant current, set a voltage limit on the power supply at, or slightly above the maximum expected voltage to avoid unsafe conditions. At constant current voltage increases as resistance increases. If a local fault condition occurs (e.g., a bad connection), high local resistance may cause the voltage to reach the maximum for the power supply, leading to overheating and damage of the electrophoresis cell.

• Resistance decreases as the temperature increases.

Voltage The velocity of an ion in an electric field varies in proportion to the field strength (volts per unit distance). The higher the voltage, the faster an ion moves. For most applications, we recommend a constant voltage setting. • A constant voltage setting allows the current and power to decrease over the course of electrophoresis, providing a safety margin in case of a break in the system. • The constant voltage setting does not need adjustment to for differences in number or thickness of gels being electrophoresed.


Mini Gel Tank

XCell SureLock Mini-Cell

XCell4 SureLock Midi-Cell

Gel capacity

Up to 2 minigels

Up to 2 minigels (8 x 8 cm)

Up to 4 midigels (8 x 13 cm)

Cell dimensions (L x W x H)

32 x 11.5 x 16 cm (height with lid on)

14 x 13 x 16 cm (height with lid on)

21 x 19 x 16 cm (height with lid on)

Advantages

• The Mini Gel Tank is versatile and compatible with NuPAGE, Bolt, or Novex minigels. The unique tank design enables convenient sideby-side gel loading and enhanced viewing during use. • Mini Blot Module is available for wet protein transfers.

• XCell II Blot Module is available for semi-wet protein transfers • Instrument incorporates a gel tension wedge in place of the rear wedge used on earlier models

• Advanced apparatus for easier, more reliable electrophoresis with midigels

Power Wattage measures the rate of energy conversion, which is manifested as heat generated by the system. Using constant power ensures that the total amount of heat generated by the system remains constant throughout the run, but results in variable mobility since voltage increases and current decreases over the course of the run. Constant power is typically used when using IEF strips. When using constant power, set the voltage limit slightly above the maximum expected for the run. High local resistance can cause a large amount of heat to be generated over a small distance, damaging the electrophoresis cell and gels.

 Learn more at thermofisher.com/electrophoresischambers



Protein standards



Protein gel stains

52

Select precast gel


Select the standard


Run the gel

Stain the gel


Post stain

53

Mini Gel Tank One tank, 181 gels The Mini Gel Tank is designed for more intuitive use and greater convenience compared to traditional electrophoresis tanks (Figure 24). The unique, side-by-side tank design allows you to perform electrophoresis of 1 or 2 minigels.

1. Snap the electrophoresis tank into the base, and place the cassette clamp(s) into the chamber(s) with the anode connector(s) (+) aligned to the center.

2. Remove the comb, and peel away the tape at the bottom of the gel cassette.

3. Place the cassette in the chamber with the wells facing towards you.

Rinse the wells 3 times with 1X buffer.

Hold the cassette in a raised position and close the clamp by moving the cam handle forward.

Fill the chamber(s) with 1X buffer to the level of the cathode.

The Mini Gel Tank offers: • Versatility—compatible with all of our minigels, including NuPAGE, Novex, Bolt, and specialty gels • Easy sample loading—forward-facing well configuration • Simultaneous visualization of both gels—streamlined, side-by-side tank configuration • Simple monitoring of gels—white tank stand provides easy visualization of prestained markers • Less running buffer required—gel chambers are separated, so you only need to load sufficient buffer for each gel to the specified fill line

Specifications • Gel capacity: up to 2 minigels • Cell size (L x W x H): 32 x 11.5 x 16 cm (height with lid on) • Buffer requirement: 400 mL for each minigel chamber • Material: polycarbonate • Chemical resistance: not compatible with acetone, chlorinated hydrocarbons, or aromatic hydrocarbons

4. Make sure the wells are completely filled with 1X buffer. Load your samples and markers.

5. Hold the cassette and release the cassette clamp.

6. Make sure the power supply is off.

Gently lower the casette so that it rests on the bottom of the chamber, and close the cassette clamp.

If only running one gel, remove the cassette clamp from unused chamber.

Add 1X buffer to the level of the fill line.

Place the lid on the tank and plug the electrode cords into the power supply. Turn the power supply on to begin electrophoresis.

Figure 24. How to use the Mini Gel Tank.

Figure 25. Electrophoresis of Bolt gel using the Mini Gel Tank. Protein standards and samples were loaded at 10 µL sample volumes in an Invitrogen™ Bolt™ 4–12% Bis-Tris Plus Gel. Electrophoresis was performed using the Mini Gel Tank at 200 V (constant). Sharp, straight bands with consistent migration patterns were observed after staining with SimplyBlue SafeStain. Images were acquired using a flatbed scanner. Lane 1: SeeBlue Plus2 Prestained Standard; Lane 2: 10 µg E. coli lysate; Lane 3: Mark12 Unstained Standard (blend of 12 purified proteins); Lane 4: 40 µg HeLa cell lysate; Lane 5: 20 µg HeLa cell lysate; Lane 6: 5 µg BSA; Lane 7: 40 µg Jurkat cell lysate; Lane 8: 5 µg GST fusion protein; Lane 9: Novex Sharp Unstained Protein Standard; Lane 10: 5 µg β-galactosidase.

Watch our Mini Gel Tank video. thermofisher.com/minigeltank

Recommended products The Mini Blot Module is a wet transfer device that conveniently fits into the chambers of the Mini Gel Tank to easily transfer proteins from minigels to nitrocellulose or PVDF membranes.

 Learn more at thermofisher.com/minigeltank For ordering information refer to page 87.



Protein standards



Protein gel stains

54

Select precast gel


Select the standard


Run the gel

Stain the gel


Post stain


55

Figure 26. How to use the XCell SureLock Mini-Cell.

Simultaneous electrophoresis of up to 2 minigels The unique design of the Invitrogen™ XCell™ SureLock Mini-Cell allows you to run minigels quickly and easily without any clamps or grease (Figure 26). The tight seal provided by the gel tension wedge results in leak-free, consistent performance. The XCell SureLock Mini-Cell is compatible with NuPAGE, Novex, and specialty gels (Figure 27).

1. Drop buffer core into the lower 2. Lock the gel tension wedge in buffer chamber of the XCell place, load samples, and fill the SureLock Mini-Cell. Insert one buffer chambers with the minigel in front of the buffer core and appropriate running buffers. a second minigel or the buffer dam behind the buffer core.

Key features of the XCell SureLock Mini-Cell:

Specifications

• -friendly design—uses single gel tension wedge with no clamps or grease

• Gel capacity: up to 2 minigels

• Flexibility—perform electrophoresis of 2 minigels simultaneously

• Buffer chamber requirement (Novex minigels):

• Unique, heat dissipating design—no need for a cooling device • Built-in safety features—retractable plugs, recessed jacks, and a specially designed lid enhances safety

• Chemical resistance: The XCell SureLock Mini-Cell is impervious to most alcohols but not compatible with acetone, chlorinated hydrocarbons (e.g., chloroform), or aromatic hydrocarbons (e.g., toluene, benzene)

Learn more at thermofisher.com/surelockmini


Figure 27. Electrophoresis of NuPAGE Bis-Tris gels with the XCell SureLock Mini-Cell. Lane 1: SeeBlue Plus2 Prestained Standard; Lane 2: 10 µg E. coli lysate; Lane 3: Mark12 Unstained Standard (blend of 12 purified proteins); Lane 4: 40 µg HeLa cell lysate; Lane 5: 20 µg HeLa cell lysate; Lane 6: not used; Lane 7: 40 µg Jurkat cell lysate; Lane 8: 5 µg of a GST fusion protein; Lane 9: Invitrogen™ Novex™ Sharp Protein Standard; and Lane 10: 5 µg β-galactosidase. Gel electrophoresis was performed at 200 V (constant) and gels were stained using SimplyBlue SafeStain. Images were acquired using a flatbed scanner.

• Cell size (L x W x H): 14 x 13 x 16 cm (height with lid on)

–– Upper buffer chamber: 200 mL –– Lower buffer chamber: 600 mL

3. Place the cell lid on the unit and you’re ready to run.

NuPAGE Bis-Tris gel in XCell SureLock Mini-Cell

Recommended products The XCell SureLock Mini-Cell can be easily adapted for transfer of proteins from minigels to membranes by simply inserting the XCell II Blot Module in the lower buffer chamber.



Protein standards



Protein gel stains

56

Select precast gel


Select the standard


Run the gel

Stain the gel


Post stain

XCell4 SureLock Midi-Cell

A.

Simultaneous electrophoresis of up to 4 midigels The Invitrogen™ XCell4 SureLock™ Midi-Cell allows simultaneous vertical electrophoresis of 1–4 midigels without leaking, enabling consistent performance. The system is designed to dissipate heat effectively and evenly, and enable highresolution results when using Novex midigels (Figure 29).

B.

1. Insert the XCell4 SureLock Assembly in its unlocked position into the center of the Midi-Cell base. The XCell4 SureLock Assembly slides down over the protrusion in the Midi-Cell base.

4. The upper buffer chamber (cathode) is the void formed between a gel and the buffer core at the center of each core.

2. Place one cassette on each side of the buffer core for each of the two cores. For each cassette, the shorter “well” side of the cassette must face out towards the lower buffer chamber.

5. Lock the XCell4 SureLock Assembly by moving the tension lever to the locked position (indicated on the XCell4 SureLock Assembly). This will squeeze the gels and buffer cores together, creating leak-free seals.

3. While holding the assembly together with your hands (A), insert the buffer cores with the gel cassettes into the lower buffer chamber such that the negative electrode fits into the opening in the gold plate on the lower buffer chamber (B). Always hold the cassette assembly by its edges as shown in the figure.

6. Proceed to loading samples and buffers.

Note: If you are having difficulty inserting the assembly into the lower buffer chamber, make sure the cathode (black polarity indicator) of the buffer core is aligned with the cathode (black polarity indicator) of the lower buffer chamber.

Specifications Key features of the XCell4 SureLock Midi-Cell: • -friendly design—leak-free electrophoresis without clamps or grease • Flexibility—perform electrophoresis of 1–4 midigels • Unique, heat dissipating design—no need for a cooling device • Built-in safety features—specially designed lid enhances safety

57

• Gel capacity: up to 4 midigels (8 x 13 cm) • Cell size (L x W x H): 21 x 19 x 16 cm (height with lid on) • Buffer chamber requirement: –– Upper buffer chamber: 175 mL x 4 –– Lower buffer chamber: 540–700 mL • Chemical resistance: not compatible with acetone, chlorinated hydrocarbons, or aromatic hydrocarbons

Figure 28. How to use the XCell4 SureLock Midi-Cell with 4 gels.

1

2

3

4

5

6

7

8

9

10 11

12

13

14

15

16

17

18

19 20

Figure 29. Quality of a precast NuPAGE Novex 4–12% Bis-Tris Midi Gel with a variety of protein standards, lysates and purified proteins. Electrophoresis was performed using MES running buffer and an XCell4 SureLock Midi Cell at 200 V (constant). Following electrophoresis, the gel was stained using SimplyBlue SafeStain, destained using water, and imaged using a flatbed scanner. Sharp, straight bands were observed. Lanes 1, 10, 11, and 20 were each loaded with 5 µL of Mark12 Unstained Standard (blend of 12 purified proteins). Lanes 2, 9, 12, and 19 were each loaded with 10 µg of E. coli lysate. Lanes 3 and 18 were each loaded with 6 µg of human IgG. Lanes 4 and 17 were each loaded with 6 µg of human IgM. Lanes 5 and 16 were each loaded with 5 µL of SeeBlue Plus2 Prestained Protein Standard. Lanes 6 and 15 were each loaded with 5 µL of BenchMark Protein Ladder. Lanes 7 and 14 were each loaded with 15 µL of MagicMark XP Western Protein Standard. Lanes 8 and 13 were each loaded with 5 µL of HiMark Unstained Protein Standard.

Learn more at thermofisher.com/surelockmidi For ordering information refer to page 87.



Protein standards



Protein gel stains

58

Select precast gel


Select the standard


Run the gel

Stain the gel

Post stain

Select precast gel



Select the standard

Stain the gel

Run the gel

Post stain

59

Run the gel PowerEase 90W Power Supply

Table 4. Gel running conditions in electrophoresis chamber systems. Running conditions in XCell Surelock Mini-Cell

Simple, affordable power supply specifically for minigel electrophoresis

Bolt 4–12% (MES)

The Invitrogen™ PowerEase™ 90W Power Supply is designed specifically for minigel electrophoresis. The straightforward, intuitive interface makes the powering of gel runs a simple and easy process. In addition, the PowerEase 90W Power Supply features: • Constant voltage or current settings • Built-in timer for walk-away gel electrophoresis • Output jacks that are compatible with most electrophoresis devices

PowerEase 300W Power Supply

Voltage (V)

Starting current (mA)*

End current (mA)*

Approximate run time (minutes)

NA

NA

NA

NA

Running conditions in Mini Gel Tank

Voltage (V)

Starting current (mA)*

Approximate End current run time (mA)* (minutes)

200

160

70

20

Bolt 4–12% (MOPS)

NA

NA

NA

NA

200

160

50

35

NuPAGE 4–12% Bis-Tris (MES)

200

100 to 125

60 to 80

35

200

160

90

30

NuPAGE 4–12% Bis-Tris (MOPS)

200

100 to 125

60 to 80

50

200

140

50

42

Novex 4–20% Tris-Glycine (denatured)

125

30 to 40

8 to 12

90

125

40

10

100

Novex 4–20% Tris-Glycine (native)

125

6 to 12

3 to 6

1 to 12 hours

125

30

10

90

NuPAGE 3–8% Tris-Acetate (denatured)

150

40 to 55

25 to 40

60

150

60

20

50

NuPAGE 3–8% Tris-Acetate (native)

150

18

7

2 to 3 hours

150

40

10

100

Novex 10–20% Tricine

125

80

40

90

125

110

40

65

NativePAGE 3–12%

150

12 to 16

2 to 4

90 to 115

150

10

<10

80

pH 3-10 IEF

10% Zymogram (Gelatin)

100

7

NA

60

100

8

NA

60

200

NA

NA

60

200

NA

NA

60

500

NA

5

30

500

NA

5

30

125

30 to 40

8 to 12

90

125

40

10

90

* Per gel. Note: Run times may vary depending on the power supply and gel percentage.

 Learn more at thermofisher.com/powerease

Programmable power supply designed for high-throughput gel electrophoresis The Invitrogen™ PowerEase™ 300W Power Supply is a fully programmable power supply designed for high-throughput gel electrophoresis. The straightforward, intuitive interface makes the powering of gel runs a simple and easy process. In addition, the PowerEase 300W Power Supply features: • Constant voltage, current, or power settings • Built-in timer for walk-away gel electrophoresis • Up to 10 custom programs with 10 steps each • Four sets of output jacks that are compatible with most electrophoresis devices




Protein standards



Protein gel stains

60

Select precast gel


Select the standard


Run the gel

Stain the gel


Post stain

61

Troubleshooting tips XCell SureLock Mini-Cell troubleshooting

Electrophoresis troubleshooting

Observation

Cause

Solution

Problem

Possible cause

Suggested solution

Run taking longer than usual

Buffers are too dilute

Check if buffer was diluted properly. Check buffer recipe; dilute from concentrate or remake if necessary.

Run taking longer time with recommended voltage

Running buffer too dilute

Make fresh running buffer and use a 1X dilution.

Upper buffer chamber is leaking

Make sure the buffer core is firmly seated, the gaskets are in place and the gel tension lever is locked.

Running buffer too concentrated

Make fresh running buffer and use a 1X dilution.

Voltage is set too low

Set correct voltage.

Current too high and excessive heat generated with recommended voltage

Remove tape from bottom of cassette.

Current too low or no current with recommended voltage

Incomplete circuit

Tape left on the bottom of the cassette Connection to power supply not complete

Check all connections with a voltmeter for conductance.

Remove the tape from the bottom of the gel cassette prior to electrophoresis. Make sure the buffer covers sample wells; check the wire connections on the buffer core.

Insufficient buffer level

Make sure the upper buffer (cathode) is covering the wells of the gel. Be sure there is sufficient buffer in the Lower Buffer Chamber to cover the slot at the bottom of the gel.

Streaking of proteins

Sample overload

Load less protein.

High salt concentration in sample

Decrease the sample salt concentration by dialysis or gel filtration.

Sample precipitates

Increase the concentration of SDS in the sample.

Contaminants such as lipids or DNA complexes in sample

Centrifuge or clarify the sample to remove particulate contaminants. Treat sample with nuclease(s).

Poorly poured gel

Make sure the gel is poured evenly and all at once.

Protein sample only partially denatured

Fully denature the protein.

Protein sample only partially reduced

Make sure a sufficient amount of DTT or β-mercaptoethanol is added.

Gel runs for too long

Watch the dye front as an indicator for proper running time.

Loading a large volume of sample causes incomplete stacking

Load appropriate volume of sample. If the sample is too dilute, concentrate it using ultrafiltration.

Uneven electric field during run

Try to make sure the loading is symmetrical if the protein concentration is known.

Uneven surface of the resolving gel

Try to make the resolving gel surface even while pouring the gel.

Expired gels

Use the gels before the specified expiration date; Note: NuPAGE gels have an extended 12 month shelf life, minimizing the risk of having expired gels.

Current reading on power supply is zero or very low

Run is faster than normal with Buffers are too concentrated poor resolution or incorrect

Cannot see the sample wells to load sample

Check buffer recipe; dilute or re-make if necessary.

Voltage, current, or wattage is set at a higher limit

Decrease power conditions to recommended running conditions (see page 59).

There is little contrast between the sample well and the rest of the gel

Mark cassette at the bottom of the wells with a marker pen prior to assembling the Upper Buffer Chamber. Illuminate the bench area with a light source placed directly behind the XCell SureLock unit.

Fuzzy bands

Mini Gel Tank troubleshooting Observation

Cause

Solution

Run taking longer than usual

Buffers are too dilute

Check buffer recipe; dilute from concentrate or remake if necessary.

Buffer chamber is leaking

Make sure the cassette clamp is firmly seated, the gaskets are in place and the cassette clamp is locked.

Current is set too low

Set correct current.

Tape left on the bottom of the cassette

Remove tape from bottom of cassette.

Connection to power supply not complete

Check all connections with a voltmeter for conductance.

Insufficient buffer level

Make sure there is sufficient buffer in the electrophoresis tank to cover the wells of the gel.

Current reading on power supply is zero or very low

Run is faster than normal with Buffers are too concentrated poor resolution or incorrect

Cannot see the sample wells to load sample

Dumbbell shaped bands or “smiling” bands

Check buffer recipe; dilute or re-make if necessary.

Current is set at a higher limit

Decrease current to recommended running conditions (see page 59).

There is little contrast between the sample well and the rest of the gel

Mark cassette at the bottom of the wells with a marker pen prior to placing the cassette in the electrophoresis tank.



Protein standards



Protein gel stains

62

Select precast gel


Select the standard


Stain the gel

Run the gel

Stain the gel

Protein stains

Silver stains

Once protein bands have been separated by electrophoresis, they can be directly visualized using different methods of in-gel detection. Over the past several decades, demands for improved sensitivity for small sample sizes and compatibility with downstream applications and detection instrumentation have driven the development of several basic staining methods. Each method has particular advantages and disadvantages, and a number of specific formulations of each type of method provide optimal performance for various situations.

• Invitrogen™ SilverXpress™ Silver Stain


Post stain

Coomassie dye protein gel stains

• Thermo Scientific™ Pierce™ Silver Stain

• Thermo Scientific™ Pierce™ Silver Stain for Mass Spectrometry

Fluorescent/specialty stains

Coomassie stains

• Pierce Reversible stain for nitrocellulose or PVDF membranes • Pro-Q™ Emerald Glycoprotein stain • Pro-Q™ Diamond Phosphoprotein stain To visualize the proteins, a protein-specific, dye-binding or color-producing chemical reaction must be performed on the

™

• SimplyBlue SafeStain • Thermo Scientific™ Imperial™ Protein Stain

The most common methods for in-gel protein detection use stains with Coomassie dye. These stains use either the G-250 (colloidal) or R-250 form of the dye (Table 6). Colloidal Coomassie stain can be formulated to effectively stain proteins within one hour and require only water (no methanol or acetic acid) for destaining.

of the stain, various steps are necessary to hold the proteins in the matrix and to facilitate the necessary chemical reaction. Most staining methods involve some version of the same general incubation steps: S

DI H2O

DI H2O

shown for SimplyBlue SafeStain (Figure 30, 33 and 34), PageBlue (Figure 31 and 35).

• Simple—Coomassie dye–based formulations are easy to formulate and are widely used

Learn more at thermofisher.com/coomassiestains

• Economical—Coomassie dye–based stain formulations are cost effective 1. Water wash.

2. Fix.

3. Water wash.

4. Stain.

5. Destain.

• A water-wash to remove electrophoresis buffers from the gel matrix • An acid or alcohol wash to condition or fix the gel to limit diffusion of protein bands from the matrix

• Flexible—useful for qualitative visualization, quantitative densitometry, and gel excision and analysis by mass spectrometry Table 6. Coomassie dye–based protein gel stains. SimplyBlue SafeStain

Imperial Protein Stain

PageBlue Protein Staining Solution

Type

G-250

R-250

G-250

Limit of detection

>7 ng

3 ng

5 ng

Time to stain (min)

12

60

60

Depending on the particular staining method, two or more of

Compatible with: PVDF membranes Nitrocellulose membranes

Yes No

Yes No

Yes No

these functions can be accomplished with one step. For example,

Reusable

No

No

Yes (up to 3x)

fix and stain in one step. Conversely, certain functions require

Mass spectrometry compatible

Yes

Yes

Yes

several steps. For example, silver staining requires both a

Color

Purple

Purple

Blue-green

Feature

Free of methanol and acetic acid

Photographs better than Coomassie G-250 dye

Free of methanol and acetic acid

Advantages

Rapid, sensitive completely non-hazardous (does not require methanol or acetic acid fixatives or destains) staining

Fast, ultrasensitive protein detection

Cost-effective option for fast, sensitive staining

• Treatment with the stain reagent to allow the dye or chemical to diffuse into the gel and bind (or react with) the proteins • Destaining to remove excess dye from the background gel matrix

staining reagent step and a developer step to produce the colored reaction product.

 Learn more at thermofisher.com/proteinstains For ordering information refer to page 87.

with simplified protocols. Example data and staining protocols are

Key features:

• Easy to use—simply soak the gel in stain solution and destain to observe protein bands

S

Our Coomassie stains provide sensitive protein detection along

Protein Staining Solution (Figure 32), and Imperial Protein Stain

proteins within the gel. Depending on the particular chemistry

a dye reagent that is formulated in an acidic buffer can effectively

• Thermo Scientific PageBlue stain ™

Convenient, ready-to-use reagents with no permanent chemical modification

• Invitrogen™ SYPRO™ Orange, Red, or Ruby gel stain

Typically these stains can be classified broadly based on the dye or molecule that helps visualize the protein stains:

63



Protein standards



Protein gel stains

64

Select precast gel


Select the standard


Run the gel

Stain the gel


Post stain

Protocols

65

Example data 1 2 3 4 5 6 7 8 9 10 2

3

1

DI H2O

S

DI H2O

DI H2O

Figure 30. SimplyBlue SafeStain protocol.

1. Wash the gel three times (5 minutes) with ultrapure water.

2. Add SimplyBlue SafeStain (1 hour).

3. Wash gel with 100 mL of DI water for 1 hour.

Figure 33. Sensitive staining results with SimplyBlue SafeStain. The following samples were separated on a NuPAGE Novex 4-12% Bis-Tris gel and then stained with SimplyBlue SafeStain. Lane 1: 6 µg protein mix; Lane 2: 1 µg rabbit IgG; Lane 3: 1 µg reduced BSA; Lane 4: 5 µg E. coli lysate; Lane 5: 20 ng reduced BSA; Lane 6: 10 ng reduced BSA; Lane 7: 7 ng reduced BSA; Lane 8: 3 ng reduced BSA; Lane 9: 10 µL Mark12 Unstained Standard (blend of 12 purified proteins); Lane 10: 5 µL Mark12 Unstained Standard.

4. Additional water wash with 100 mL of DI water (1 hour) for increased sensitivity.

1

3 4

5

6

7

8

9

1

2

3 4

5

6

7

8

9

1

2

3 4

5

6

7

8

9

Rabbit IgG

S

DI H2O

2

Figure 34. Two-dimensional electrophoresis (2DE) analysis of spinach chloroplast extract; staining with SimplyBlue SafeStain. Spinach chloroplast extract was prefractionated in the ZOOM™ IEF Fractionator and the individual fractions were then separated by 2DE using narrow pH range ZOOM™ Strips and NuPAGE™ Novex 4–12% Bis-Tris ZOOM™ Gels. Gels were Coomassie stained using SimplyBlue SafeStain.

DI H2O BSA Protein A

Figure 31. Imperial Protein Stain protocol.

1. Wash the gel three times with deionized water (15 minutes).

2. Add Imperial Protein Stain (5 minutes−1 hour).

3. Water destain (15 minutes−overnight).

Protein G Lysozyme

5-minute stain; 15-minute water destain

1-hour stain; 2-hour water destain

1-hour stain; overnight water destain

Figure 35. Enhanced sensitivity and clear background using Imperial Protein Stain. For even greater sensitivity and reduced background, gels can be stained with Imperial Protein Stain for 1 hour and washed with water from 1 hour to overnight. Lane 1: BSA only (6 µg); Lane 2–9: loaded left to right with 1,000, 200, 100, 50, 25, 12, 6, and 3 ng protein sample.

Did you know? DI H2O

S

DI H2O

Staining with a Coomassie stain prior to silver staining allows for more uniform staining of certain proteins since silver ions can interact with certain functional groups such as carboxylic acid groups, imidazole, sulfhydryls, and amines.

DI H2O


1. Wash the gel three times with ultrapure water (30 minutes).

2. Add PageBlue Protein Staining Solution (1 hour).


3. Rinse gel two times with ultrapure water (<1 minute)

4. Wash gel one time with ultrapure water (5 minutes)

Figure 32. PageBlue Protein Staining Solution protocol.

The Thermo Scientific Pierce Power Stainer is designed for rapid Coomassie dye staining of proteins in up to two minigels and subsequent removal of unbound stain from the gel in a single step. Refer to page 72 of this brochure for more information.



Protein standards



Protein gel stains

66

Select precast gel


Select the standard


Run the gel

Stain the gel

Silver stains

We offer highly sensitive silver stains with short protocol times that are also compatible with mass spectrometry (Table 7). The SilverXpress™ Silver Staining Kit provides nanogram-level sensitivity

Pierce Silver Stain for Mass Spectrometry

Pierce Silver Stain Kit

SilverXpress Silver Staining Kit

Components (steps)

6 (17)

4 (15)

5 (13)

Time required

1 hr 13 min

2 hr 25 min

2 hr

Limit of detection

0.25 ng

0.25 ng

0.86 ng

Mass spectrometry compatible

Yes

Yes

Yes

Storage

Room temperature

Room temperature

4°C

Stability

1 year

1 year

6 months

Advantages

• Fast and sensitive staining and destaining of protein gels • Optimized for peptide recovery after in-gel typsin digestion for mass spectrometry • Flexible gel fixation (15–30 min to overnight) and staining (1–30 min)

• Rapid, ultrasensitive and versatile silver stain system • Flexible gel fixation (30 min to overnight) and staining (5 min to 20 hours)

• Nanogram-level sensitivity for silver staining with minimal background

Protocols and example data

with minimal background (Figure 37), while the Pierce™ Silver Stain Kit provides protocol flexibility (Figure 38 and 39).

SZ

F

H2O

1 2 3 4 5 6 7 8 9 10

Key features: • Sensitive—silver stains are highly sensitive stains that allow for visualization of proteins at sub-nanogram levels

Learn more at thermofisher.com/silverstains 1. Wash the gel with water.

• Easy to use and flexible—optimized for minimal steps and flexibility to accommodate shorter or longer protocols • Workflow compatible—our mild chemical formulations help ensure compatibility with mass spectrometry and sequencing • Robust performance—detailed protocol enables consistent results with clear background

67

Table 7. Silver stain kits.

Ultra-sensitive stains with optimized protocols, manufactured for minimal variability Silver staining is the most sensitive colorimetric method for detecting total protein, and functions by the deposition of metallic silver at the location of protein bands. Silver ions (from silver nitrate in the stain reagent) interact and bind with certain protein functional groups. The strongest interactions occur with carboxylic acid groups (Asp and Glu), imidazole (His), sulfhydryl groups (Cys), and amines (Lys). Various sensitizer and enhancer reagents are essential for controlling the specificity and efficiency of silver ion binding to proteins and effective conversion (development) of the bound silver to metallic silver.


Post stain

2. Fix the gel in Fixing Solution for 10 minutes.

3. Decant the Fixing Solution and incubate the gel in 2 changes of Sensitizing Solution. S

H2 O S 4. Decant the Sensitizing Solution and rinse the gel two times with ultrapure water.

5. Incubate the gel in Staining Solution.

D

H2O

D 6. Decant the Staining Solution and rinse the gel two times with ultrapure water.

7. Incubate the gel in Developing Solution.

SS

Figure 37. Crystal clear background with the SilverXpress Kit. Samples were separated on a NuPAGE Novex 4-12% Bis-Tris gel and stained with the SilverXpress Kit. Lanes 1, 10: Mark12 Unstained Standard (blend of 12 purified proteins) diluted 1:4; Lane 2: Mark12 Unstained Standard diluted 1:8; Lane 3: Mark12 Unstained Standard diluted 1:16; Lane 4: Mark12 Unstained Standard diluted 1:32; Lane 5: Mark12 Unstained Standard diluted 1:64; Lane 6: 1.6 ng BSA; Lane 7: 0.8 ng BSA; Lane 8: E. coli lysate diluted 1:20; Lane 9: E. coli lysate diluted 1:80.

H 2O

8. Add the Stopping Solution directly to the gel when the desired staining intensity is reached.

9. Decant the Stopping Solution and wash the gel three times with ultrapure water.

Figure 36. SilverXpress Silver Staining Kit protocol.




Protein standards



Protein gel stains

68


Select precast gel

Select the standard

Acetic Acid

EtOH H2O


Run the gel

Lysate staining

Stain the gel

MW marker staining

F

2. Fix 2 x 15 minutes in EtOH/ acetic acid.

SZ

10% EtOH

SZ 3. Incubate 2 x 5 minutes with 10% EtOH. Wash.

4. Mix Sensitizer. Sensitize for 1 minute. Wash 2 x 1 minute.

Fluorescent gel stains are designed for use in 1D and 2D PAGE and offer sensitivities similar to that obtained with silver staining techniques. Invitrogen™ SYPRO™ protein stains are easy-to-use fluorescent stains for the detection of proteins separated by PAGE (Table 8). Stained proteins can be viewed with a standard UV or blue-light transilluminator or with a laser scanner.

thermofisher.com/fluorescentstains Recommended products For optimal sensitivity with Polaroid™ film, SYPRO™ Photographic Filter is recommended.

• Simple—no destaining or timed steps required; minimal hands-on time

5. Mix Silver Stain. Stain for 5 minutes. Wash 2 x 20 seconds.

5% Acetic Acid

• Quantitative—linear quantitation range over two orders of magnitude with low protein-to-protein variability • Highly sensitive—typically more sensitive than Coomassie dye–based stains and equivalent to silver stains

D 7. Remove developer. Stop with 5% Acetic Acid for 10 minutes.

Table 8. SYPRO protein stains.

Figure 38. Pierce Silver Stain Kit protocol.


Learn more at

Features:

S

6. Mix Developer. Develop for 2-3 minutes.

2 minutes, 30 seconds

Development Time

Figure 39. Pierce Silver Stain Kit exhibits excellent senstivity. In standard minigels, proteins are detectable at greater than 0.25 ng per band or spot.

S

D

2 minutes, 30 seconds

69

Fluorescent protein gel stains Rapid, highly sensitive fluorescent stains for total protein detection after electrophoresis

F 1. Wash 2 x 5 minutes with ultrapure water.


Post stain

SYPRO Ruby stain

SYPRO Orange stain

SYPRO Red stain

Limit of detection

0.25 ng

4–8 ng

4–8 ng

Stain and destain time

90 min microwave; 18 hr standard

~1 hr

~1 hr

Ex/Em

280 nm, 450/610 nm

300 nm, 470/510 nm

300 nm, 550/630 nm

Ease of use

Ready to use

Supplied as stock solution

Supplied as stock solution

Compatible applications

Mass spectrometry, IEF, 2D gels, on-membrane staining





Protein standards



Protein gel stains

70

Select precast gel


Select the standard


Run the gel

Stain the gel


Post stain

71

Stain the gel

Specialty protein stains

Electrophoretic staining technology—Pierce Power Stainer

Our specialty protein stains include in-gel phosphoprotein and glycoprotein detection and on-membrane reversible protein staining kits (Table 9).

Learn more at thermofisher.com/specialtystains

Table 9. Specialty protein stains. Pro-Q Emerald 488 Glycoprotein Gel and Blot Stain Kit

Pro-Q Emerald 300 Glycoprotein Gel and Blot Stain Kit

Pro-Q Diamond Phosphoprotein Gel Staining Kit

Detects

Glycoproteins

Glycoproteins

Phosphoproteins

Sensitivity

4 ng glycoprotein per band

0.5 ng glycoprotein per band

1–16 ng phosphoprotein per band

Stain and destain time

~6 hr

~5 hr

4–5 hr

Ex/Em

510/520 nm

280/530 nm

555/580 nm

Advantages

Selective staining of glycoproteins

Selective staining of glycoproteins

Selective staining of phosphoproteins

The Thermo Scientific Pierce Power Stainer consists of a Thermo Scientific™ Pierce™ Power Station with activated Staining Software and a Thermo Scientific™ Pierce™ Power Stain Cassette. It is designed for rapid Coomassie staining and destaining of proteins in polyacrylamide gels. Traditional Coomassie staining techniques require one hour to overnight staining and destaining to achieve desired results. When used in conjunction with Thermo Scientific™ Pierce™ Midi and Mini Gel Power Staining Kits, the Pierce Power Stainer is designed to provide staining efficiency in as few as 6 minutes that is equivalent to, or better than, traditional Coomassie staining techniques.

Good to know

How does electrostaining work? Cathode (–)

Coomassie staining pad Pre-run and pre-washed SDS-PAGE gel Destaining pad

Anode (+)

Pierce Power Stain Cassette Cathode (–) Staining pad Gel Destaining pad Anode (+)

The significant reduction in protein staining time is accomplished by utilizing inonic Power Stain Solution and Destain Solution to electrophoretically drive the negatively charged Coomassie R-250 dye out of the top gel pad, through the polyacrylamide gel matrix and the bottom gel pad, and toward the positively charged anode.

Watch our Pierce Power Stainer video. thermofisher.com/powerstainer




Protein standards



Protein gel stains

72

Select precast gel


Select the standard


Run the gel

Stain the gel


Post stain

73

Pierce Power Stainer Rapid Coomassie dye staining and destaining in approximately 10 minutes The Thermo Scientific™ Pierce™ Power Stainer is designed for rapid Coomassie dye staining of proteins in polyacrylamide gels and subsequent removal of unbound stains to give sharply stained protein bands with minimal or no background. The Pierce Power Stainer offers:

Specifications

• Speed—Coomassie dye staining and destaining of proteins in about 10 minutes

• Mode of transfer: semi-dry blotting

• Convenience—simultaneously stain and destain 1–2 minigels or 1 midigel • Reliable performance—enables staining results that are equivalent to traditional staining techniques • Easy touch programming—intuitive LCD touch-screen interface includes preprogrammed protocols


• Gel compatibility: SDS-PAGE gels

The Pierce Power System can be used both for fast Coomassie dye staining of protein gels and for rapid semi-dry transfer of proteins from gel to membrane. The Pierce Power Stainer can be upgraded by adding the Pierce™ Power Blot Cassette to make a fully functional Pierce Power System with blotting and staining capabilities.

• Running dimension: horizontal • Platform: Pierce™ Power System

Pierce Power Stainer

Conventional Coomassie stain

Did you know? Conventional Coomassie dye–based staining techniques require 1 hour to overnight incubation.

Total time: 11 minutes 1. Wash gel 1 × 5 minutes in water 2. Power Stain/Destain gel, 6 minutes

Total time: 230 minutes to overnight 1. Wash gel 3 × 10 minutes in water 2. Incubate gel in Coomassie stain solution* for 60 minutes 3. Wash gel 2 × 10 minutes in water 4. Destain gel in destaining solution** for 3 × 20 minutes 5. Incubate gel in water for 60 minutes to overnight

Thermo Scientific Pierce Power Stainer

Thermo Scientific Pierce Power Blotter

*Coomassie stain solution: 45% methanol, 10% acetic acid, 0.25% Coomassie R-250 **Destain solution: 30% ethanol, 5% acetic acid

Figure 40. Pierce Power Stainer saves time and maintains sensitivity.

Learn more at thermofisher.com/powerstainer For ordering information refer to page 87.



Protein standards



Protein gel stains

74

Select precast gel


Select the standard


Run the gel

Stain the gel

Protein gel electrophoresis technical handbook 75 75

Post stain

Western blotting Transfer and Detection

After electrophoresis, the separated proteins are transferred or blotted onto a second matrix, generally a nitrocellulose or polyvinylidene difluoride (PVDF) membrane. Next, the membrane is blocked to minimize potential nonspecific binding of antibodies to the surface of the membrane. Detailed procedures vary widely for the detection steps of the western blot workflow. One common variation involves direct vs. indirect detection methods. In both the direct and indirect detection methods, the blocked membrane is probed with an antibody (primary antibody) specific to the protein of interest (antigen). In direct detection techniques, this antibody is enzyme conjugated or labeled with a fluorophore. However, in indirect detection techniques, the blocked membrane is probed first with an antibody (primary antibody) which is specific to the antigen followed by another antibody (secondary antibody) raised against the host species of the primary antibody. This secondary antibody is often enzyme conjugated or labeled with a fluorophore. The direct method is not widely used as most researchers prefer the indirect detection method for a variety of reasons.

Horseradish peroxidase (HRP) or alkaline phosphatase (AP) are the most popular enzymes conjugated to antibodies used in the western blot workflow. After incubating the membrane with the detection antibody or antibodies, if an enzyme-conjugated antibody was utilized, an appropriate substrate (chromogenic or chemiluminescent) is added and that results in a detectable product. A popular substrate of choice is a chemiluminescent substrate that, when combined with the enzyme, produces light as a byproduct. With the chemiluminescent substrate, the light output can be captured on film or CCD camera. In recent years fluorescent detection became a popular alternative to the enzymatic detection since it allows for more quantitative data analysis. Fluorescent detection utilizes dye-labeled primary antibodies or dyelabeled secondary antibodies and the signal output is captured on an appropriate imaging system. Whatever substrate is used, the intensity of the signal should correlate with the abundance of the antigen on the blotting membrane.

Key products for western blot transfer: Wet

Semi-dry

Dry

Mini Blot Module

Thermo Scientific Pierce Power Blotter

iBlot™ 2 Dry Blotting System

Key products for western blot detection include: Automated detection

Manual detection Blocking buffers Wash buffers Detergents Enhancers Substrates Stripping buffers X-ray film

We offer a wide range of reagents, kits, equipment, and antibodies to facilitate every step of western blot analysis. iBind™ Flex Western Device

 Learn more at thermofisher.com/western



Protein standards



Protein gel stains

76


Appendix

77

Protocol quick reference QUICK REFERENCE

QUICK REFERENCE

NuPAGE Bis-Tris Midi Gels

Bolt™ Mini Gels

Bolt™ Mini Gels

Instructions for performing electrophoresis using Bolt ™ Mini Gels are described below.

Prepare gel 1. Cut open the gel cassette pouch and remove the cassette. 2. Remove the gel comb and rinse wells 3 times with 1X Running Buffer. and tank

Prepare samples

Reagent

3. Remove the tape covering the slot at the lower portion of the cassette.

Reduced Sample Non-reduced Sample

Sample Bolt ™ LDS Sample Buffer (4X) Bolt ™ Reducing Agent (10X) Deionized Water

x µL 10 µL 4 µL to 26 µL

x µL 10 µL — to 30 µL

Load samples

Total Volume 40 µL* 40 µL* Heat samples at 70˚C for 10 minutes. * Scale samples up or down by adjusting all volumes proportionally.

Prepare 1X Buffer Run conditions

Each chamber of the tank requires 400 mL of 1X SDS Running Buffer (mix 20 mL of 20X Bolt ™ MES or MOPS SDS Running Buffer with 380 mL of deionized water). The same buffer type must be used for both chambers.

1–4

Run Bolt™ Mini Gels at constant voltage (1 or 2 mini gels). Running Buffer

Standard Run

Run Time*

MES

200 V

22 min

MOPS

200 V

32 min

Prepare Samples

5–7

Cathode

QUICK REFERENCE

NuPAGE Tris-Acetate Mini Gels

NuPAGE Bis-Tris Mini Gels Reduced Sampl e

Sam pl e NuPAGE LDS Sample Buffer (4X) NuPAGE Reducing Agent (10X) Deionized Water Tot al Vol u m e

x μL 2.5 μL　 1 μL to 6.5 μL 10 μ L

Non- r educed Sampl e

Load Buffer

Run

10 μ L

Conditions

Add 50 mL 20X NuPAGE MES or MOPS SDS Running Buffer to 950 mL deionized water to prepare 1X SDS Running Buffer. Load the appropriate concentration of your protein sample on the gel. Fill the Upper (200 mL) and Lower (600 mL) Buffer Chambers with the appropriate 1X Running Buffer. For reduced samples, use 200 mL 1X Running Buffer with 500 μL NuPAGE Antioxidant in the Upper Buffer Chamber. Voltage: Run Time: Expected Current:

Prepare Samples

x μL 2.5 μL -to 7.5 μL

Heat samples at 70˚C for 10 minutes.

Load Sample

Denaturing Sample x µL 2.5 µL — 1 µL to 10 µL final

Native Sample x µL — 5 µL — to 10 µL final

*For reduced samples only.

Load Sample

Load the appropriate concentration of your protein sample on the gel.

Add Buffer

Fill Upper Buffer Chamber with 175 mL 1X NuPAGE SDS Running Buffer. For reduced samples, use 175 mL 1X NuPAGE SDS Running Buffer with 435 μL NuPAGE Antioxidant in the Upper Buffer Chamber. Add a sufficient volume of 1X NuPAGE SDS Running Buffer to the Lower Buffer Chamber.

Run

Voltage: Run Time: Expected Current:

200 V constant 40 min (MES Buffer), 55 min (MOPS Buffer) 160–200 mA/gel (start); 120–170 mA/gel (end)

Prepare 1X Buffer

Denaturing samples: Add 50 mL 20X NuPAGE Tris-Acetate SDS Running Buffer to 950 mL deionized water. Native samples: Add 100 mL 10X Tris-Glycine Native Running Buffer to 900 mL deionized water.

Load Sample Load the appropriate concentration of your protein sample on the gel. Add Buffer

Fill Upper Buffer Chamber with 175 mL of the appropriate 1X Running Buffer. For reduced samples, use 175 mL 1X Running Buffer with 435 μL NuPAGE Antioxidant in the Upper Buffer Chamber. Add a sufficient volume of Running Buffer the Lower Buffer Chamber.

Run Conditions


150 V constant 70 min (denaturing gel), 2–3 hours (native gel) 70–90 mA/gel (start); 50–60 mA/gel (end); (denaturing gel) 40–45 mA/gel (start); 15–20 mA/gel (end); (native gel)

QUICK REFERENCE

Instructions for electrophoresis using the XCell SureLock Mini-Cell are described below.

Prepare 1X Buffer

Sample NuPAGE LDS Sample Buffer (4X) Tris-Glycine Native Sample Buffer (2X) NuPAGE Reducing Agent (10X)* Deionized Water

Heat denaturing samples at 70˚C for 10 minutes. Do not heat native samples.

Add 50 mL 20X NuPAGE MES or MOPS SDS Running Buffer to 950 mL deionized water to prepare 1X SDS Running Buffer.

Conditions

Reagent

For Research Use Only. Not for use in diagnostic procedures.

For research use only. Not for use in diagnostic procedures.

Reagent

x µL 2.5 µL 1 µL to 10 µL final

Non-reduced sample x µL 2.5 µL — to 10 µL final

Prepare 1X Buffer

* Run times may vary depending upon gel type and power supply.

Prepare Samples

Reduced sample

Prepare Samples


Fill Line

Cathode

Reagent Sample NuPAGE LDS Sample Buffer (4X) NuPAGE Reducing Agent (10X) Deionized Water

1. Pre-fill the chamber with 1X Running Buffer to the level of the cathode. 2. Place the cassette in the chamber with the wells facing towards you. Hold the cassette in a raised position and close the cassette clamp. 3. Fill all wells with 1X Running Buffer. 4. Load your samples and markers. 5. Hold the cassette and release the cassette clamp. 6. Gently lower the cassette to the bottom of the chamber, and close the cassette clamp 7. Add 1X buffer to the level of the fill line.

NuPAGE Tris-Acetate Midi Gels

Instructions for electrophoresis of Bis-Tris Gels using the XCell4 SureLock Midi-Cell are described below.

200 V constant 35 minutes (MES Buffer), 50 minutes (MOPS Buffer) 100–125 mA/gel (start); 60–80 mA/gel (end)

Intended Use: For research use only. Not for human or animal therapeutic or diagnostic use.

Prepare 1X Buffer

Reagent Denatur i ng Sampl e* Nati ve Sampl e Sam pl e x μL x μL NuPAGE LDS Sample Buffer (4X) 2.5 μL -Tris-Glycine Native Sample Buffer (2X) -5 μL D ei on i z ed Water t o 7.5 μ L to 5 μL Tot al Vol u m e 10 μ L 10 μ L Samples Heat samples at 70˚C for 10 minutes Do not heat *For reduced samples, add NuPAGE Reducing Agent (10X) to 1X.

Novex Tris-Glycine Mini Gels

Novex Tris-Glycine Mini Gels

Instructions are provided below for electrophoresis of NovexTris-Glycine Gels using the XCellSureLock Mini-Cell.

Non-Denaturing (Native) Electrophoresis

Denaturing Electrophoresis

Prepare Samples

Reagent Sampl e Sample x μL Tris-Glycine Native Sample Buffer (2X) 5 μL Deionized Water to 5 μL Total Volume 10 μL Do not heat samples for native electrophoresis.

Prepare 1X Buffer

Add 100 mL 10X Tris-Glycine Native Running Buffer to 900 mL deionized water to prepare 1X Tris-Glycine Native Running Buffer.

Load Sample


Load Buffer

Fill the Upper Buffer Chamber with 200 mL and the Lower Buffer Chamber with 600 mL of 1X Tris-Glycine Native Running Buffer.

Run


Prepare Samples

Denaturing Samples: Add 50 mL 20X NuPAGE Tris-Acetate SDS Running Buffer to 950 mL deionized water. Native Samples: Add 100 mL 10X TrisGlycine Native Running Buffer to 900 mL deionized water.

Reagent Reduced Sample Sample x μL Tris-Glycine SDS Sample Buffer (2X) 5 μL NuPAGE Reducing Agent (10X) 1 μL Deionized Water to 4 μL Total Volume 10 μL

Non-reduced Sample x μL 5 μL -to 5 μL 10 μL


Load Sample


Prepare 1X Buffer

Load Buffer

Fill the Upper (200 mL) and Lower (600 mL) Buffer Chambers with the appropriate 1X Running Buffer. For reduced samples, use 200 mL 1X Running Buffer with 500 μL NuPAGE Antioxidant in the Upper Buffer Chamber.

Load Sample


Load Buffer

Fill the Upper Buffer Chamber with 200 mL and the Lower Buffer Chamber with 600 mL of 1X Tris-Glycine SDS Running Buffer.

Conditions

Run


Blot Gel

Run Conditions


150 V constant 1 hour (Denaturing gel), 2–3 hours (Native gel) 40–55 mA/gel (start); 25–40 mA/gel (end) for denaturing gel 18 mA/gel (start); 7 mA/gel (end) for native gel

Conditions

Add 100 mL 10X Novex Tris-Glycine SDS Running Buffer to 900 mL deionized water to prepare 1X Tris-Glycine SDS Running Buffer.

125 V constant 90 minutes (dependent on gel percentage) 30–40 mA/gel (start); 8–12 mA/gel (end)

125 V constant 1–12 hours 6–12 mA/gel (start); 3–6 mA/gel (end)

For blotting denaturing and native gels, use 1X Tris-Glycine Transfer Buffer with 20% methanol. Perform blotting at 25 V constant for 1–2 hours using the XCell II Blot Module. The expected start current is 100 mA.

Intended Use: For research use only. Not for human or animal therapeutic or diagnostic use.

Appendix

78


Appendix

79

Protocol quick reference QUICK REFERENCE

QUICK REFERENCE

Tris-Glycine Midi Gels

Non-denaturing (Native) Electrophoresis

Instructions for electrophoresis using the XCell4 SureLock Midi-Cell are described below.

Prepare Samples

Reagent

Reduced sample

Sample Tris-Glycine SDS Sample Buffer (2X) NuPAGE Reducing Agent (10X) Deionized Water

x µL 5 µL 1 µL to 10 µL final

Non-reduced sample x µL 5 µL — to 10 µL final


Prepare 1X Buffer

Add 100 mL 10X Tris-Glycine SDS Running Buffer to 900 mL deionized water to prepare 1X Tris-Glycine SDS Running Buffer.

Load Sample


Add Buffer

Fill each Upper Buffer Chamber with 175 mL 1X Tris-Glycine SDS Running Buffer. Fill the Lower Buffer Chamber up to the fill line mark with 1X Tris-Glycine SDS Running Buffer.

Run Conditions


Prepare Samples

Reagent Sample Tris-Glycine Native Sample Buffer (2X) Deionized Water

Native Sample x µL 5 µL to 10 µL final

Do not heat native samples.

Prepare 1X Buffer

Add 100 mL 10X Tris-Glycine SDS Running Buffer to 900 mL deionized water to prepare 1X Tris-Glycine SDS Running Buffer.

Load Sample


Add Buffer

Fill each Upper Buffer Chamber with 175 mL of 1X Tris-Glycine Native Running Buffer. Fill the Lower Buffer Chamber up to the fill line mark with 1X TrisGlycine Native Running Buffer.

Run Conditions


125 V constant 1–12 hours 35–40 mA/gel (start); 15–20 mA/gel

Tricine Gels

Tricine Gels

Instructions are provided below for electrophoresis of Tricine Gels using the XCell SureLock Mini-Cell.

Blotting Conditions

For blotting Tricine gels, use 1X Tris-Glycine Transfer Buffer with 20% methanol. Perform transfer with nitrocellulose or PVDF membranes at 25 V constant for 1–2 hours using the XCell II Blot Module. The expected start current is 100 mA.

Alternate Transfer Buffers

The Tris-Glycine Transfer Buffer interferes with protein sequencing. If you are performing protein sequencing, use 1X NuPAGE Transfer Buffer or 0.5X TBE Transfer Buffer for blotting. The NuPAGE Transfer Buffer protects against modification of the amino acid side chains and is compatible with N-terminal protein sequencing using Edman degradation.

Prepare Samples

Reagent Reduced Sampl e Sam pl e x μL Tricine SDS Sample Buffer (2X) 5 μL NuPAGE Reducing Agent (10X) 1 μL Deionized Water to 4 μL Tot al Vol u m e 10 μ L

Non- r educed Sampl e x μL 5 μL -to 5 μL 10 μ L


Prepare 1X Buffer

Add 100 mL 10X Novex Tricine SDS Running Buffer to 900 mL deionized water to prepare 1X Tricine SDS Running Buffer.

Load Sample Load the appropriate concentration of your protein sample on the gel. Load Buffer Fill the Upper Buffer Chamber with 200 mL and the Lower Buffer Chamber

125 V constant 105 min (dependent on gel percentage) 40–50 mA/gel (start); 20–25 mA/gel (end)

with 600 mL of 1X Tricine SDS Running Buffer.

Run Conditions



125 V constant 90 minutes (dependent on gel percentage) 80 mA/gel (start); 40 mA/gel (end)

For research use only. Not for human or animal therapeutic or diagnostic use.

QUICK REFERENCE

QUICK REFERENCE

Staining Protocol

NativePAGE Bis-Tris Gels ™

Instructions are provided below for electrophoresis of NativePAGE™ Bis-Tris Gels using the XCell SureLock Mini-Cell.

Prepare Samples

Reagent Sample NativePAGE™ Sample Buffer (4X) NativePAGE™ 5% G-250 Additive Deionized Water

Sample with detergent x μL 2.5 μL 0.25–1 μL* to 10 μL

Detergent-free sample x μL 2.5 μL — to 10 μL

Do not heat samples for native gel electrophoresis. *Ensure the final G-250 concentration is ¼th the detergent concentration.

Prepare 1X Running Buffer

1X NativePAGE Anode Buffer: Add 50 mL 20X NativePAGE Running Buffer to 950 mL deionized water. 1X NativePAGE™ Cathode Buffer: Add 50 mL 20X NativePAGE™ Running Buffer and 50 mL 20X NativePAGE™ Cathode Additive to 900 mL deionized water. ™

™

Load Sample Fill wells with 1X NativePAGE™ Cathode Buffer and load samples prior to filling

the cathode chamber. Load an appropriate amount of your sample on the gel.

Add Buffer Run Conditions

A quick staining protocol for NativePAGE Gels using the Coomassie G-250 from the sample additive is described below. The total staining time is ~2–3 hours. Sensitivity is ~60 ng BSA. Step

1 2

Action

Time

Place the gel in 100 mL fixing solution (40% methanol, 10% acetic acid) and microwave on high (950–1100 watts).

45 seconds

Shake the gel on an orbital shaker.

15 minutes

3

Discard fixing solution.

4

Place the gel in 100 mL destain solution (8% acetic acid) and microwave on high (950–1100 watts).

5

Shake the gel on an orbital shaker until the desired background is obtained.

IEF Gels

IEF Gels

Instructions are provided below for electrophoresis of IEF Gels using the XCell SureLock Mini-Cell.

Prepare for 1. Stain and destain the IEF gel. Incubate the IEF gel in 100 mL 20% ethanol 2D SDS/ for 10 minutes. PAGE 2. Cut out the desired lane (strip) from the gel for transfer to a SDS gel.

Prepare Samples

— 45 seconds

Reagent Sample IEF Sample Buffer pH 3–10 or pH 3–7 (2X) Deionized Water

Prepare 1X Buffer

1X IEF Anode Buffer: Add 20 mL 50X IEF Anode Buffer to 980 mL deionized water. Chill to 4°C to 10°C. 1X IEF Cathode Buffer: Add 20 mL IEF Cathode Buffer pH 3–10 (10X) or pH 3–7 (10X) to 180 mL deionized water. Chill to 4°C to 10°C.

Load Sample

Load the appropriate concentration and volume of your protein on the gel.

Add Buffer

Fill the Upper Buffer Chamber with chilled 200 mL 1X IEF Cathode Buffer and the Lower Buffer Chamber with chilled 600 mL 1X IEF Anode Buffer.

Run

Voltage: Expected Current:

—

Conditions

100 V constant for 1 hour 200 V constant for 1 hour 500 V constant for 30 minutes 7 mA/gel (start); 5 mA/gel (end)

Fill the Upper Buffer Chamber with ~200 mL 1X NativePAGE™ Cathode Buffer. Fill the Lower Buffer Chamber with ~550 mL 1X NativePAGE™ Anode Buffer.

Stain Gel



150 V constant 90–115 min (3–12% gel); 105–120 min (4–16% gel) 12–16 mA/gel (start); 2–4 mA/gel (end)

Sample x μL 5 μL to 10 μL final

Fix the IEF gel in 12% TCA or 12% TCA containing 3.5% sulfosalicylic acid for 30 minutes. Stain the IEF gel with method of choice.

3. Incubate the gel strip in 2 mL 2X SDS sample buffer and 0.5 mL ethanol for 3–5 minutes. Aspirate the sample buffer and rinse the gel strip with 1X SDS Running Buffer. 4. Fill the SDS gel cassette with 1X SDS Running Buffer. 5. Trim the IEF gel strip to a length of 5.8–5.9 cm. 6a. Transfer the gel strip into a 1.0 mm SDS gel by sliding the strip into the 2D-well using a gel loading tip. Avoid trapping air-bubbles between the strip and the SDS gel. Wet a piece of thick filter paper (5.8 cm × 4 cm) in SDS Running Buffer and insert the long edge of the paper into the 2D-well to push the gel strip into with the SDS gel. 6b. If transferring the gel strip into a 1.5 mm SDS gel, wet 2 pieces of thin filter paper (5.8 cm × 4 cm) in 1X SDS Running Buffer. Sandwich the gel strip between the two filter papers along the long edge with the edge of the strip protruding ~0.5 mm beyond the paper. Insert the sandwich into the 2D-well of the SDS gel without trapping air bubbles and push the strip down so it is in with the SDS gel. 7. Insert gel into the mini-cell, fill the buffer chambers with 1X SDS Running Buffer, and perform SDS-PAGE. 8. After the dye front has moved into the stacking gel (~10 minutes), disconnect power, remove the paper, and resume electrophoresis.


Appendix

80


Appendix

81

Ordering information QUICK REFERENCE

Zymogram Gels

Zymogram Gels

Instructions are provided below for electrophoresis of Zymogram Gels using the XCell SureLock Mini-Cell.

Develop Gel

Prepare Samples

Prepare 1X Buffer

Reagent Sample Sample x μL Tris-Glycine SDS Sample Buffer (2X) 5 μL Deionized Water to 10 μL final Do not heat or reduce samples for Zymogram gels. 1X Tris-Glycine SDS Running Buffer: Add 100 mL 10X Tris-Glycine SDS Running Buffer to 900 mL deionized water.

Load Sample

Load the appropriate concentration and volume of your protein on the gel.

Add Buffer

Fill the Upper Buffer Chamber with 200 mL, and the Lower Buffer Chamber with 600 mL of 1X Tris-Glycine SDS Running Buffer.

Run Conditions


125 V constant 90 minutes (dependent on gel percentage) 30–40 mA/gel (start); 8–12 mA/gel (end)


Stain Gel

Sensitivity Level

Product

1. Dilute Zymogram Renaturing Buffer (10X) and Zymogram Developing Buffer (10X) 1:9 with deionized water. Prepare 100 mL of each 1X buffer per 1–2 mini-gels. 2. After electrophoresis, incubate the gel in 1X Zymogram Renaturing Buffer for 30 minutes at room temperature with gentle agitation. 3. Decant Zymogram Renaturing Buffer and add 1X Zymogram Developing Buffer to the gel. 4. Equilibrate the gel for 30 minutes at room temperature with gentle agitation. 5. Decant buffer and add fresh 1X Zymogram Developing Buffer to the gel. 6. Incubate the gel at 37°C for at least 4 hours or overnight for maximum sensitivity. Optimize results empirically by varying the sample load or incubation time. Zymogram (Blue Casein) 4–16% gels do not require staining. For non-prestained Zymogram gels, stain the gels with Colloidal Blue Staining Kit or the SimplyBlue Safestain. Areas of protease activity appear as clear bands against a dark background. 10% Zymogram (Gelatin) Gel: 12% Zymogram (Casein) Gel: 4–16% Zymogram (Blue Casein) Gel:

10-6 units of collagenase 7 × 10-4 units of trypsin 1.5 × 10-3 units of trypsin

Quantity

Cat. No.

Product

Quantity

Cat. No.

NuPAGE™ Novex™ 10% Bis-Tris Protein Gels, 1.0 mm, 9-well

1 box

NP0307BOX


1 box

NP0315BOX


1 box

NP0316BOX


1 box

NP0344BOX


10 gels

NP0341BOX


2 gels

NP0341PK2


10 gels

NP0342BOX


2 gels

NP0342PK2


1 box

NP0343BOX


1 box

NP0349BOX

1 box

NP0324BOX

NW3010

NuPAGE™ Novex™ 4–12% Bis-Tris Protein Gels, 1.0 mm, 1-well

10 gels

NP0321BOX

NW3012

NuPAGE™ Novex™ 4–12% Bis-Tris Protein Gels, 1.0 mm, 10-well NuPAGE™ Novex™ 4–12% Bis-Tris Protein Gels, 1.0 mm, 10-well

2 gels

NP0321PK2


10 gels

NP0322BOX


2 gels

NP0322PK2


10 gels

NP0323BOX

Select precast gel Bolt Bis-Tris Plus Gels Bolt 8% Bis-Tris Plus Gels, 10-well

1 box

NW00080BOX

Bolt™ 8% Bis-Tris Plus Gels, 12-well

1 box

NW00082BOX


1 box

NW00085BOX


1 box

NW00087BOX


1 box

NW00100BOX

Bolt 10% Bis-Tris Plus Gels, 12-well

1 box

NW00102BOX


1 box

NW00105BOX


1 box

NW00107BOX


1 box

NW00120BOX


1 box

NW00122BOX


1 box

NW00125BOX


1 box

NW00127BOX

Bolt 4–12% Bis-Tris Plus Gels, 10-well

1 box

NW04120BOX


1 box

NW04122BOX

Bolt™ 4–12% Bis-Tris Plus Gels, 15-well

1 box

NW04125BOX


1 box

NW04127BOX

Bolt™ Empty Mini Gel Cassettes

20 cassettes NW2010

Bolt Empty Mini Gel Cassette Combs, 10-well

20 combs

Bolt™ Empty Mini Gel Cassette Combs, 12-well

20 combs

Bolt Welcome Pack B, 4–12%, 15-well

1 kit**

NW0412B

Bolt™ Welcome Pack A, 4–12%, 10-well

1 kit**

NW0412A

™

™

™

™

™

™

™

™

™

™

One box contains 10 gels. ** B olt Welcome Pack kit includes Mini Gel Tank, 2 boxes of Bolt 4–12% Gels (10-well/15-well), Bolt MES Running Buffer (20X), Bolt LDS Sample Buffer (4X), Bolt Sample Reducing Agent (10X) and SeeBlue™ Plus2 Prestained Standard

NuPAGE Bis-Tris Mini Gels (8 cm x 8 cm) NuPAGE™ Novex™ 10% Bis-Tris Protein Gels, 1.0 mm, 1-well

1 box

NP0304BOX


2 gels

NP0323PK2


10 gels

NP0301BOX


10 gels

NP0329BOX


2 gels

NP0301PK2


2 gels

NP0329PK2


10 gels

NP0302BOX


1 box

NP0327BOX


2 gels

NP0302PK2


10 gels

NP0335BOX


1 box

NP0303BOX


2 gels

NP0335PK2

Appendix

82


Appendix

83

Ordering information Product

Quantity

Cat. No.

Product

Quantity

Cat. No.

Product

Quantity

Cat. No.

Product

Quantity

Cat. No.


10 gels

NP0336BOX

NuPAGE™ Novex™ 3–8% Tris-Acetate Protein Gels, 1.0 mm, 10-well

2 gels

EA0375PK2

Novex™ 10% Tris-Glycine Mini Protein Gels, 1.5 mm, 10-well

1 box

EC6078BOX


1 box

EC6505BOX


2 gels

NP0336PK2


10 gels

EA03752BOX


1 box

EC60785BOX


1 box

EC65052BOX

2 gels

EA03752PK2

Novex™ 10–20% Tris-Glycine Mini Protein Gels, 1.0 mm, 10-well

1 box

EC6135BOX


1 box

EC65055BOX

WG1201BOX


1 box

EA03755BOX


1 box

EC61352BOX


1 box

EC6508BOX

WG1201A


1 box

EA0378BOX


1 box

EC61355BOX


1 box

EC65085BOX

WG1202BOX


1 box

EA03785BOX


1 box

EC61385BOX


1 box

EC6055BOX

WG1202A


1 box

EA0355BOX


1 box

EC6001BOX


1 box

EC60552BOX

WG1203BOX

NuPAGE™ Novex™ 7% Tris-Acetate Protein Gels, 1.0 mm, 10-well

1 box

EA03552BOX


1 box

EC6005BOX


1 box

EC6058BOX

WG1203A


1 box

EA03555BOX


1 box

EC60052BOX


1 box

EC60585BOX

WG1401BOX


1 box

EA0358BOX


1 box

EC60055BOX


1 box

EC6035BOX

WG1401A


1 box

EA03585BOX


1 box

EC6008BOX


1 box

EC60352BOX

WG1402BOX


1 box

EC60085BOX


1 box

EC60355BOX

WG1601BOX


1 box

EC6485BOX


1 box

EC6038BOX

WG1601A


1 box

EC64852BOX


1 box

EC60385BOX

WG1602BOX


1 box

EC64855BOX


1 box

EC6021BOX

WG1602A


1 box

EC6488BOX


1 box

EC6025BOX

WG1603BOX


1 box

EC64885BOX


1 box

EC60252BOX

WG1603A

Novex™ 14% Tris-Glycine Mini Protein Gels, 1.5 mm, 15-well Novex™ 16% Tris-Glycine Mini Protein Gels, 1.0 mm, 10-well

1 box

EC6495BOX


1 box

EC60255BOX

NuPAGE Bis-Tris Midi Gels (8 cm x 13 cm) NuPAGE Novex 10% Bis-Tris Midi Protein Gels, 12+2-well

1 box

NuPAGE Novex 10% Bis-Tris Midi Protein Gels, 12+2-well, w/adapters

1 box

NuPAGE™ Novex™ 10% Bis-Tris Midi Protein Gels, 20-well

1 box

NuPAGE Novex 10% Bis-Tris Midi Protein Gels, 20-well, w/adapters

1 box

NuPAGE Novex 10% Bis-Tris Midi Protein Gels, 26-well

1 box

NuPAGE™ Novex™ 10% Bis-Tris Midi Protein Gels, 26-well, w/adapters

1 box

NuPAGE Novex 4–12% Bis-Tris Midi Protein Gels, 12+2-well

1 box

NuPAGE Novex 4–12% Bis-Tris Midi Protein Gels, 12+2-well, w/adapters

1 box

NuPAGE Novex 4–12% Bis-Tris Midi Protein Gels, 20-well

1 box

NuPAGE Novex 4–12% Bis-Tris Midi Protein Gels, 20-well, w/adapters

1 box

WG1402A

NuPAGE Novex 3–8% Tris-Acetate Midi Protein Gels, 12+2W

1 box

NuPAGE Novex 4–12% Bis-Tris Midi Protein Gels, 26-well

1 box

WG1403BOX

NuPAGE Novex 3–8% Tris-Acetate Midi Protein Gels, 12+2W, w/adapters

1 box

™

NuPAGE Novex 4–12% Bis-Tris Midi Protein Gels, 26-well, w/adapters

1 box

WG1403A

NuPAGE Novex 3–8% Tris-Acetate Midi Protein Gels, 20W

1 box

NuPAGE Novex 8% Bis-Tris Midi Protein Gels, 12+2-well

1 box

WG1001BOX

NuPAGE Novex 3–8% Tris-Acetate Midi Protein Gels, 20W, w/adapters

1 box

™

NuPAGE Novex 8% Bis-Tris Midi Protein Gels, 12+2-well, w/adapters

1 box

WG1001A

NuPAGE Novex 3–8% Tris-Acetate Midi Protein Gels, 26W

1 box


1 box

WG1002BOX

NuPAGE Novex 3–8% Tris-Acetate Midi Protein Gels, 26W, w/adapters

1 box

NuPAGE™ Novex™ 8% Bis-Tris Midi Protein Gels, 20-well, w/adapters

1 box


1 box

NuPAGE Novex 8% Bis-Tris Midi Protein Gels, 26-well, w/adapters

1 box

™

™

™

™

™

™

™

™

™

™

™

™

™

™

™

™

™

™

™

™

™

™

™

™

™

™

™

™

NuPAGE Tris-Acetate Midi Gels (8 cm x 8 cm)

10 gels

™

™

™

™

™

™

™

™

™

™

™

WG1002A

Novex Tris-Glycine Mini Gels (8 cm x 8 cm) 1 box

EC6075BOX


1 box

EC64952BOX


1 box

EC60249BOX

WG1003BOX


3 boxes

EC6075BX30


1 box

EC64955BOX


1 box

EC6028BOX

WG1003A

Novex™ 10% Tris-Glycine Mini Protein Gels, 1.0 mm, 10-well - Value Pack Novex™ 10% Tris-Glycine Mini Protein Gels, 1.0 mm, 12-well

1 box

EC60752BOX


1 box

EC6498BOX


1 box

EC60285BOX


1 box

EC60755BOX


1 box

EC64985BOX


1 box

EC6065BOX

NuPAGE Tris-Acetate Mini Gels (8 cm x 8 cm) NuPAGE™ Novex™ 3–8% Tris-Acetate Protein Gels, 1.0 mm, 10-well

™

EA0375BOX

Appendix

84


Appendix

85


Quantity

Cat. No.

Product

Quantity

Cat. No.

Product


1 box

EC60652BOX

Novex™ 4–20% Tris-Glycine Midi Protein Gels, 12+2-well, w/adapters

1 box

WT4201A

Novex Tricine Gels 1 box

EC6675BOX

Pierce™ SDS-PAGE Sample Prep Kit

1 box

Novex 4–20% Tris-Glycine Midi Protein Gels, 20-well

1 box

WT4202BOX

50 reactions

89888

Novex 6% Tris-Glycine Mini Protein Gels, 1.0 mm, 15-well

Novex 10% Tricine Protein Gels, 1.0 mm, 10-well

1 box

EC66752BOX

Bolt Transfer Buffer (20X)

125 mL

BT0006

1 box

WT4202A

Bolt Transfer Buffer (20X)

1L

BT00061

WT0101BOX

Novex 4–20% Tris-Glycine Midi Protein Gels, 20-well, w/adapters

Novex™ 10% Tricine Protein Gels, 1.0 mm, 12-well

1 box

EC6695BOX

4X Bolt LDS Sample Buffer

10 mL

B0007

1 box

WT4203BOX

WT0101A

Novex™ 4–20% Tris-Glycine Midi Protein Gels, 26-well


1 box

EC66952BOX

20X Bolt MES SDS Running Buffer

500 mL

B0002

1 box

WT4203A

WT0102BOX

Novex™ 4–20% Tris-Glycine Midi Protein Gels, 26-well, w/adapters


1 box

WT0102A

Novex™ 8% Tris-Glycine Midi Protein Gels, 12+2-well

1 box

WT0103BOX

Novex™ 8% Tris-Glycine Midi Protein Gels, 12+2-well, w/adapters

1 box

WT0103A

Novex™ 8% Tris-Glycine Midi Protein Gels, 20-well

1 box

WT0121BOX

Novex™ 8% Tris-Glycine Midi Protein Gels, 20-well, w/adapters

1 box

WT0083BOX

Novex™ pH 3–7 IEF Protein Gels, 1.0 mm, 12-well

5 gels⁄box

WT0121A


1 box

WT0083A

WT0122BOX



1 box

WT8161BOX

WT0122A

Novex™ 8–16% Tris-Glycine Midi Protein Gels, 12+2-well


1 box

WT8161A

Novex Zymogram Gels

WT0123BOX

Novex™ 8–16% Tris-Glycine Midi Protein Gels, 12+2-well, w/adapters

1 box

WT0123A


1 box

WT4121BOX


1 box

WT4121A

Novex™ 8–16% Tris-Glycine Midi Protein Gels, 26-well Novex™ 8–16% Tris-Glycine Midi Protein Gels, 26-well, w/adapters

1 box

™

EC60655BOX

Novex Tris-Glycine Midi Gels (8 cm x 13 cm) Novex™ 10% Tris-Glycine Midi Protein Gels, 12+2-well

1 box

Novex™ 10% Tris-Glycine Midi Protein Gels, 12+2-well, w/adapters

1 box


1 box

Novex 10% Tris-Glycine Midi Protein Gels, 20-well, w/adapters

1 box


1 box


1 box

Novex 12% Tris-Glycine Midi Protein Gels, 12+2-well

1 box

Novex 12% Tris-Glycine Midi Protein Gels, 12+2-well, w/adapters

1 box

Novex 12% Tris-Glycine Midi Protein Gels, 20-well

1 box


1 box

Novex 12% Tris-Glycine Midi Protein Gels, 26-well

1 box


1 box

Novex 4–12% Tris-Glycine Midi Protein Gels, 12+2-well

1 box

Novex 4–12% Tris-Glycine Midi Protein Gels, 12+2-well, w/adapters

1 box

™

™

™

™

™

™

™

™

™

™

™

™

WT4122BOX


1 box

WT4122A

NativePAGE™ Novex™ 3–12% Bis-Tris Protein Gels, 1.0 mm, 10-well

1 box


1 box

WT4123BOX


1 box

Novex 4–12% Tris-Glycine Midi Protein Gels, 26-well, w/adapters

1 box

NativePAGE Novex 4–16% Bis-Tris Protein Gels, 1.0 mm, 10-well

1 box

Novex™ 4–20% Tris-Glycine Midi Protein Gels, 12+2-well

1 box


1 box

WT4123A WT4201BOX

Quantity

Cat. No.

EC66955BOX

20X Bolt MES SDS Running Buffer

5L

B0002-02

20X Bolt MOPS SDS Running Buffer

500 mL

B0001

20X Bolt MOPS SDS Running Buffer

5L

B0001-02

1 box

WT0081BOX

Novex 10–20% Tricine Protein Gels, 1.0 mm, 10-well

1 box

EC6625BOX

Bolt Antioxidant

15 mL

BT0005

WT0081A

LA0041

1 box

EC66252BOX

NuPAGE Tris-Acetate SDS Running Buffer (20X)

500 mL

WT0082BOX

Novex™ 10–20% Tricine Protein Gels, 1.0 mm, 12-well

NP0001

1 box

EC66255BOX

NuPAGE™ MOPS SDS Running Buffer (20X)

500 mL

WT0082A

Novex™ 10–20% Tricine Protein Gels, 1.0 mm, 15-well

NuPAGE™ MOPS SDS Running Buffer (20X)

5L

NP000102

EC66452BOX

NuPAGE™ MES SDS Running Buffer (20X)

5L

NP000202

5 gels⁄box

EC6645BOX

NuPAGE™ MES SDS Running Buffer (20X)

500 mL

NP0002

5 gels⁄box

EC6655BOX

Novex Tris-Glycine SDS Running Buffer (10X)

4x1L

LC26754 LC2675

1 box

EC64052BOX


500 mL

WT8162BOX

Novex™ 12% Zymogram (Casein) Protein Gels, 1.0 mm, 12-well

LC26755

1 box

EC6415BOX


5L

WT8162A

Novex™ 4–16% Zymogram (Blue Casein) Protein Gels, 1.0 mm, 10-well

LC1675

1 box

EC6405BOX

Novex™ Tricine SDS Running Buffer (10X)

500 mL

WT8163BOX

Novex™ 12% Zymogram (Casein) Protein Gels, 1.0 mm, 10-well

NuPAGE™ LDS Sample Buffer (4X)

10 mL

NP0007

1 box

EC61755BOX

WT8163A

Novex™ 10% Zymogram (Gelatin) Protein Gels, 1.0 mm, 15-well

Novex Tricine SDS Sample Buffer (2X)

20 mL

LC1676

20 mL

LC2676

™


1 box

EC61752BOX

Novex Tris-Glycine SDS Sample Buffer (2X)

BN1001BOX


1 box

EC6175BOX

Novex™ Tris-Glycine Transfer Buffer (25X)

500 mL

LC3675

BN1003BOX

E-PAGE™ High Throughput Gel System

NuPAGE™ Transfer Buffer (20X)

125 mL

NP0006

E-PAGE™ 8% Protein Gels, 48-well

EP04808

NuPAGE Transfer Buffer (20X)

1L

NP00061

EH03

NuPAGE Antioxidant

15 mL

NP0005

EPBUF01

Novex Tris-Glycine SDS Buffer Kit

1 kit

LC2677

NuPAGE MOPS SDS Buffer Kit (for Bis-Tris Gels)

1 kit

NP0050

NuPAGE™ MES SDS Buffer Kit (for Bis-Tris Gels)

1 kit

NP0060

NativePAGE Gels

™

Product

Prepare samples and select buffers: SDS-PAGE

Novex IEF Gels

1 box

™

Cat. No.

Novex™ 16% Tricine Protein Gels, 1.0 mm, 15-well ™


™

Quantity

BN1002BOX BN1004BOX

E-Holder™ Platform E-PAGE™ Loading Buffer 1

8 gels 2 units 4.5 mL

E-PAGE™ 6% Protein Gels, 96-well

8 gels

EP09606

Daughter E-Base™ Device

1 unit

EBD03

Mother E-Base Device

1 unit

EBM03

™

™

™

™

™

Appendix

86


Appendix

87


Quantity

Cat. No.

Product

NuPAGE™ Tris-Acetate SDS Buffer Kit (for Tris-Acetate gels), Contains 1 ea. LA0041, NP0004, NP0005, NP0007

1 kit

LA0050

Select Protein Standards: Unstained

Novex™ Tricine SDS Buffer Kit, includes LC1676 & LC1675

1 kit

Pierce LDS Sample Buffer, NonReducing (4X) Pierce Lane Marker Non-Reducing Sample Buffer

5 mL 5 mL

LC1677 84788 39001

Pierce 10X Tris-Glycine SDS Buffer

1L

28362

BupH Tris-Glycine-SDS Buffer Packs

40 packs

28378

™

Native Electrophoresis Novex Tris-Glycine Native Running Buffer (10X)

500 mL

LC2672

Novex Tris-Glycine Native Sample Buffer (2X)

20 mL

LC2673

NativePAGE™ Running Buffer (20X)

1L

BN2001

NativePAGE™ Running Buffer Kit

1 kit

BN2007

NativePAGE™ Cathode Buffer Additive (20X)

250 mL

BN2002

NativePAGE™ Sample Buffer (4X)

Quantity

Cat. No.

PageRuler™ Unstained Low Range Protein Ladder

2 x 250 µL

26632

PageRuler™ Unstained Protein Ladder

2 x 250 µL

26614


5 x 50 µL

LC0725

Prestained

BN2003

Mini Gel Tank

1 unit

A25977

PageBlue Protein Staining Solution

1L

24620


1 unit

EI0001

SimplyBlue SafeStain

1L

LC6060

XCell4 SureLock™ Midi-Cell

1 each

WR0100

SimplyBlue SafeStain

3.5 L

LC6065

PowerEase™ 90W Power Supply (115 VAC)

1 each

PS0090


1L

24615


3x1L

24617


1 each

PS0091

Pierce™ Silver Stain Kit

1L

24612

SilverXpress™ Silver Staining Kit

1 kit*

LC6100

Pierce™ Silver Stain for Mass Spectrometry

1L

24600

SYPRO™ Orange Protein Gel Stain

500 µL

S-6650

SYPRO™ Orange Protein Gel Stain

10 x 50 µL

S-6651

™

™

2 x 250 µL

26619


1 each

PS0300

PageRuler™ Plus Prestained Protein Ladder

10 x 250 µL

26620


1 each

PS0301

Spectra™ Multicolor Broad Range Protein Ladder

2 x 250 µL

26634

Spectra™ Multicolor Broad Range Protein Ladder

10 x 250 µL

26623

HiMark Prestained Protein Standard

250 µL

LC5699

SYPRO™ Red Protein Gel Stain

500 µL

S-6653


2 x 250 µL

26625

SYPRO™ Red Protein Gel Stain

10 x 50 µL

S-6654

SYPRO Ruby Protein Gel Stain

1L

S-12000


200 mL

S-12001


5L

S-21900

Pro-Q Emerald 488 Glycoprotein Gel Stain Kit

1 kit

P-21875

Pro-Q™ Diamond Phosophoprotein Gel Stain Kit

1L

P-33300


200 mL

P-33301


5L

P-33302


1 unit

22833

Pierce Power Stainer Welcome Pack

1 unit

22833SPCL*

™

™

Silver stains

*1 kit contains sufficient reagents to stain 25 mini gels

Fluorescent and specialty stains

™

™

MagicMark™ XP Western Protein Standard

250 µL

LC5602

DDM (n-dodecyl β-D-maltoside) (10%)

1 mL

BN2005

Specialty

1 mL

BN2006


2 x 250 µL

500 mL

LC2671

BenchMark™ Fluorescent Protein Standard

125 µL

LC2670


125 µL

LC5606

IEF Marker 3–10

500 µL

39212-01

™

™

IEF

Coomassie stains

PageRuler™ Plus Prestained Protein Ladder

BN2008

500 mL


26617

1 kit

Novex™ Zymogram Renaturing Buffer (10X)

50 µL

LC5603

26635 LC5928

™

™


™

Novex™ IEF Anode Buffer (50X)

100 mL

LC5300

Novex™ IEF Cathode Buffer pH 3-10 (10X) 125 mL

LC5310

Novex™ IEF Cathode Buffer pH 3-7 (10X)

125 mL

LC5370

Novex pH 3-10 IEF Buffer Kit, Includes LC5300, LC5310, LC5311

1 kit

LC5317

Novex™ pH 3-7 IEF Buffer Kit, Includes LC5300, LC5370, LC5371

1 kit

LC5377

Novex™ IEF Sample Buffer pH 3-10 (2X)

25 mL

LC5311

Novex IEF Sample Buffer pH 3-7 (2X)

25 mL

LC5371

™

™

Cat. No.

10 x 250 µL

NativePAGE Sample Prep Kit

Novex™ Zymogram Developing Buffer (10X)

Quantity

PageRuler™ Prestained Protein Ladder

MagicMark™ XP Western Protein Standard

Zymography

Product

26616

0.5 mL

Digitonin (5%)

Cat. No.

2 x 250 µL

NativePAGE™ 5% G-250 Sample Additive ™

BN2004

Quantity

PageRuler™ Prestained Protein Ladder

Western 10 mL

Product

*Welcome pack includes Pierce Power Station, Pierce Power Stain Cassette, Western Blot Roller, Power Cord with C/13 Connector, Quick Start Guide, Pierce Mini Gel Power Staining Kit

Appendix Appendix

Crossword puzzle challenge To participate in the crossword puzzle challenge, Separate Crossword

go to thermofisher.com/pagecrossword Complete the crossword below 1

2

3 4 5

6

7 8

9

10 11

12

13

14

15

Across

Created on TheTeachersCorner.net Crossword Maker

Xcell4SureLock

DownHermannSchagger UlrichLaemmli ArneTiselius

SheldonEngelhorn

5. Can you name one of the scientists who developed 1. Which tank is compatible with >180 mini gels? pg. 52 BisTris MagicMark PowerStainer Bolt minimizes Minigeltank HarrySvennsonRilbe blue native polyacrylamide gel electrophoresis? 2. Which gel chemistry protein degradation? pg. 9 pg. 17Unstained 6. Which tank is compatible with midigels? pg. 56 3. Who first published SDS-PAGE as a method for the Prestained PowerEase NuPAGE 9. Which ladder can be used for accurate molecular analysis of cleavage of structural proteins in weight estimation directly on western blots? pg. 46 bacteriophage? pg. 7 10. Can you name one of the scientists who filed a 4. Can you name one of the scientists who first patent for the netural-pH Bis-Tris system in 1996? pg. 11 described the theory of separation of amphoteric 12. Which gel has a unique wedge well design that proteins along a pH gradient in the 1960s? pg. 21 allows you to load 2x the sample volume? pg. 10 7. What power supply is available for use with the Mini 13. Which protein ladder would you use for Gel Tank? pg. 58 approximate determination of molecular weight? pg. 35 8. What is a fast alternative to traditional Coomassie 15. Who won the Nobel Prize for analysis of serum staining? pg. 72 proteins by electrophoresis in 1948? pg. 5 11. Which ladder would you use for precise determination of molecular weight? pg. 35 14. Which type of gel has been referenced in >20,000 publications? pg. 12

Learn more at thermofisher.com/invitrogen For Research Use Only. Not for use in diagnostic procedures. © 2015 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Polaroid is a trademark of Polaroid Corporation. Triton is a trademark of Union Carbide Corporaton. CO015381 0715

Protein Gel Electrophoresis Technical Handbook 114311

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