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Criterion™Precast GelApplication Guide

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Table of Contents

Section 1 General Information.............................................................................................. 11.1 Introduction........................................................................................................................................... 11.2 Criterion System Specifications............................................................................................................. 21.3 Criterion Comb Configurations.............................................................................................................. 2

Section 2 Setup and Basic Operation.................................................................................. 32.1 Setting Up and Running Criterion Gels................................................................................................. 32.2 Opening Criterion Cassettes and Removing the Gel............................................................................. 4

Section 3 SDS-PAGE............................................................................................................. 53.1 Introduction.......................................................................................................................................... 53.2 Criterion Tris-HCl Gel Composition....................................................................................................... 63.3 Criterion Tris-HCl Gel Selection Guide.................................................................................................. 63.4 SDS-PAGE Buffers............................................................................................................................... 73.5 Sample Preparation.............................................................................................................................. 73.6 Running Conditions.............................................................................................................................. 7

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Section 4 Native PAGE......................................................................................................... 84.1 Introduction........................................................................................................................................ 84.2 Criterion Tris-HCl Gel Composition..................................................................................................... 84.3 Criterion Tris-HCl Gel Selection Guide ............................................................................................... 94.4 Native PAGE Buffers............................................................................................................................ 94.5 Sample Preparation............................................................................................................................ 104.6 Running Conditions ........................................................................................................................... 10

Section 5 Peptide Analysis............................................................................................................... 115.1 Introduction........................................................................................................................................ 115.2 Criterion Tris-Tricine/Peptide Gel Composition .................................................................................... 115.3 Criterion Tris-Tricine/Peptide Gel Selection Guide ............................................................................... 115.4 Tris-Tricine/Peptide Buffers.................................................................................................................. 125.5 Sample Preparation............................................................................................................................ 125.6 Running Conditions............................................................................................................................ 12

Section 6 Isoelectric Focusing......................................................................................................... 136.1 Introduction........................................................................................................................................ 136.2 Criterion IEF Gel Composition ............................................................................................................ 136.3 Criterion IEF Gel Selection Guide ....................................................................................................... 136.4 IEF Buffers.......................................................................................................................................... 146.5 Sample Preparation............................................................................................................................ 146.6 Running Conditions............................................................................................................................ 14

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Section 7 Protease Analysis by Zymogram PAGE................................................................ 157.1 Introduction......................................................................................................................................... 157.2 Criterion Zymogram Composition........................................................................................................ 157.3 Criterion Zymogram Selection Guide....................................................................................................157.4 Zymogram Buffers............................................................................................................................... 167.5 Sample Preparation............................................................................................................................. 167.6 Running Conditions............................................................................................................................. 16

Section 8 Nondenaturing Nucleic Acid PAGE.......................................................................178.1 Introduction......................................................................................................................................... 178.2 Criterion TBE Gel Composition............................................................................................................ 178.3 Criterion TBE Gel Selection Guide....................................................................................................... 178.4 Nondenaturing Nucleic Acid PAGE Buffers...........................................................................................188.5 Sample Preparation............................................................................................................................. 188.6 Running Conditions............................................................................................................................. 18

Section 9 Denaturing Nucleic Acid PAGE.............................................................................199.1 Introduction..........................................................................................................................................199.2 Criterion TBE-Urea Gel Composition....................................................................................................199.3 Criterion TBE-Urea Gel Selection Guide...............................................................................................199.4 TBE-Urea Buffers.................................................................................................................................209.5 Sample Preparation............................................................................................................................. 209.6 Running Conditions............................................................................................................................. 20

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Section 10 Detection........................................................................................................... 2110.1 SDS-PAGE and Native PAGE Detection....................................................................................... 21–2210.2 Peptide Gel Staining......................................................................................................................... 2310.3 IEF Gel Staining................................................................................................................................ 2410.4 Zymogram Gel Staining.................................................................................................................... 2510.5 TBE Gel Staining.............................................................................................................................. 2610.6 TBE Urea Gel Staining...................................................................................................................... 26

Section 11 Troubleshooting............................................................................................27–28

Section 12 Ordering Information....................................................................................29–3312.1 Criterion Gels............................................................................................................................... 29–3012.2 Criterion Gel Accessories................................................................................................................. 3112.3 Buffers............................................................................................................................................. 3112.4 Detection Reagents.......................................................................................................................... 3212.5 Blotting Membranes......................................................................................................................... 3312.6 Protein and DNA Standards............................................................................................................. 3312.7 Equipment....................................................................................................................................... 33

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Section 1General Information1.1 Introduction

Criterion is the next generation of dedicated precast gel systems. The innovative, easy-to-use designproduces superior resolution while running more samples per gel. Compared to any other precast gelsystem, Criterion produces more results while providing significant cost and time savings. Some of the newand unique features provided are:

• Patented integral buffer chamber that eliminates all buffer leaks• Up to 26 sample capacity per gel• Flexibility to run one or two gels• Multichannel pipet compatible gels• Outlined and numbered wells that simplify sample loading• J-foot (patent pending) that improves gel drying and blotting results

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1.2 Criterion System Specifications

Gel material Polyacrylamide

Gel dimensions 8.7 x 13.3 cm

Gel thickness 1.0 mm

Resolving gel height 6.5 cm

Cassette dimensions 10.6 x 15.0 cm

Cassette material Styrene copolymer

Comb material Polycarbonate

Storage tray material PET

Upper running buffer volume 60 ml

Lower running buffer volume 800 ml

Storage conditions Store flat at 4ºC; DO NOT FREEZE

1.3 Criterion Comb Configurations

Comb Load Volume Comments

12+2 well 45 µl with two 15 µl reference wells Multichannel pipet compatible18-well 30 µl 26-well 15 µl Multichannel pipet compatiblePrep+2 well 800 µl with two 15 µl reference wellsIPG 11 cm ReadyStrip IPG stripIPG+1 well 11 cm ReadyStrip IPG strip with one 15 µl reference well

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Section 2Setup and Basic Operation2.1 Setting Up and Running Criterion Gels

1. Each Criterion gel is packaged individually in a plastic storage tray. Remove the cover by gently pulling thesquare corner tab up and diagonally across the package. Remove the gel from the package.

2. Remove the comb and gently rinse the wells with dd H2O or running buffer.

3. Remove the tape from the bottom of the cassette by pulling the tab across the gel.

4. Insert the Criterion gel into one of the slots in the Criterion cell tank.Ensure that each integral buffer chamber faces the center of thecell.

5. Fill each integral buffer chamber with 60 ml running buffer.

6. Load samples using a Hamilton syringe or a pipet with gel loadingtips. A sample loading guide can be placed on the outer edge ofthe cassette, to aid in aligning pipet tips with the wells. This isespecially useful with multichannel pipets.

7. Fill each half of the lower buffer tank with 400 ml of running buffer to the marked fill line.

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8. Place the lid on the tank, aligning the color-coded banana plugs and jacks. See sections 3–9 for powerconditions.

2.2 Opening Criterion Cassettes and Removing the Gel

1. After electrophoresis is complete, turn off the power supplyand disconnect the electrical leads.

2. Remove the lid from the tank, and remove the Criterion gel(s)from the cell. Pour off and discard the upper running buffer.

3. Invert the cassette and place the integral buffer chamber overthe cassette-opening tool built into the Criterion cell lid.

4. Firmly press down on the cassette to crack the cassette weldson both sides of the cassette. The cassette will split openapproximately 1/3 of the way.

5. Alternatively, the gel cassette can be opened by sliding thetapered back of the comb into the slits on either side of thecassette.

6. Pull the two halves of the cassette apart to completely exposethe gel.

7. Remove the gel by either floating the gel into a fixing or stainingsolution or by carefully lifting the gel from the cassette.

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Section 3SDS-PAGE3.1 Introduction

Criterion Tris-HCl gels provide a versatile system for the separation of proteins by molecular weight (SDS-PAGEconditions) or charge to mass ratio (native conditions). (See section 4 for native PAGE applications andprotocols.) This versatility is possible because Criterion Tris-HCl gels are made without SDS, allowing thesample buffer and running buffer to determine the separation mechanism. Historically, SDS-PAGE systemscontained SDS in both the gel and the running buffer. Reproducible SDS-PAGE separations are performed ingels lacking SDS, provided the sample buffer and running buffers contain sufficient SDS to saturate theproteins during electrophoresis. The recommended concentration of SDS is 2% in sample buffer and 0.1% inrunning buffers.

SDS-PAGE uses discontinuous chloride and glycine ion fronts to form moving boundaries that stack and then separate SDS coated polypeptides by molecular weight. Protein samples are prepared in a reducingdenaturing sample buffer containing either 2-mercaptoethanol or dithiothreitol as the reducing reagent, and heat and SDS are used to denature the proteins. 2-Mercaptoethanol and dithiothreitol eliminate proteinsecondary structure by reducing disulfide bonds. SDS minimizes charge variability among proteins, givingthem the same charge to mass ratio and forcing them into rod-like shapes. This effectively eliminates theeffects of protein conformation and native charge density on the electrophoretic migration distance. Molecularweight determinations are obtained by plotting the logarithm of protein molecular mass vs. the relativemobility (Rf = distance migrated by protein/distance migrated by dye front).

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3.2 Criterion Tris-HCl Gel Composition

Gel buffer 0.375 M Tris-HCl, pH 8.6Cross-linker 2.6% CStacking gel 4% T, 2.6% CStorage buffer 0.375 M Tris-HCl, pH 8.6Shelf life ~12 weeks; individual expiration date is printed on each cassette

3.3 Criterion Tris-HCl Gel Selection Guide

Tris-HCl gels are available in a wide selection of single percentages and gradients for the separation ofproteins by SDS-PAGE.

Tris-HCl Gels Optimal Separation5% 100–250 kD7.5% 40–200 kD10% 30–150 kD12.5% 20–120 kD15% 10–100 kD18% 10–50 kD

Tris-HCl Gradient Gels Optimal Separation4–15% 20–250 kD4–20% 10–200 kD8–16% 20–12 kD10–20% 10–100 kD10.5–14% 25–200 kD

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3.4 SDS-PAGE Buffers

Running buffer, 1x 25 mM Tris192 mM glycine0.1% SDSDO NOT ADJUST pH

Sample buffer 62.5 mM Tris-HCl, pH 6.82% SDS25% glycerol0.01% Bromophenol Blue5% 2-mercaptoethanol or 350 mM DTT (added fresh)

3.5 Sample Preparation

Determine the appropriate protein concentration of your sample based on the detection method and loadvolume used. (See section 10.1 for approximate stain sensitivities.) Dilute 1 part sample with 2 parts samplebuffer (see section 3.4) and heat at 95ºC for 5 min.

3.6 Running Conditions

Power conditions 200 V constantStarting current: 90–120 mA/gelFinal current: 35–55 mA/gel

Run time 55 min

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Section 4Native PAGE4.1 Introduction

Criterion Tris-HCl gels are made without SDS, allowing separation of protein in their native conformation. Thenonreducing and nondenaturing environment of native PAGE allows the detection of biological activity andcan improve antibody detection. Native PAGE can also be used to resolve multiple protein bands wheremolecular mass separation by SDS-PAGE would reveal only one.

Native PAGE uses the same discontinuous chloride and glycine ion fronts as SDS-PAGE to form movingboundaries that stack and then separate polypeptides by charge to mass ratio. Proteins are prepared in a nonreducing, nondenaturing sample buffer that maintains the proteins’ secondary structure and native chargedensity. Native PAGE is not suitable for accurate molecular weight determination due to the variability ofcharge to mass ratio among different proteins.

4.2 Criterion Tris-HCl Composition


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4.3 Criterion Tris-HCl Selection

Native PAGE separates by charge to mass ratio, making individual protein migration protein dependent.Optimal Tris-HCl gel percentages will have to be determined experimentally.

4.4 Native PAGE Buffers

Running buffer, 1x 25 mM Tris192 mM glycineDO NOT ADJUST pH

Sample buffer 62.5 mM Tris-HCl, pH 6.825% glycerol0.01% Bromophenol Blue

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Determine the desired protein concentration and load volume of your sample based on the detection methodused. (See section 10.1 for approximate stain sensitivities.) Sample preparation for native PAGE applicationsrequires special consideration. In the absence of SDS, the net charge of a polypeptide will be determined bythe pH of the sample buffer. Only polypeptides with a net negative charge will migrate into a native PAGECriterion Tris-HCl gel. Most polypeptides have an acidic or slightly basic pl (~3–8). These proteins can beseparated using a standard protocol by diluting 1 part sample with 2 parts native sample buffer (see section4.4; DO NOT HEAT SAMPLES).

Strongly basic peptides (pl >9) will have a net positive charge in a native PAGE Criterion Tris-HCl gel. In orderfor polypeptides with a net positive charge to migrate into a native PAGE Criterion Tris-HCl gel, the polarity ofthe electrodes must be changed by reversing the color-coded jacks when connecting to the power supply.



Run time 55 min

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Section 5 Peptide Analysis5.1 Introduction

Criterion Tris-Tricine/peptide gels are optimized for separating peptides and proteins with molecular weight<10,000. Superior resolution of peptides is achieved by moving the peptide-SDS complexes more slowlythrough the gel. This allows the faster moving SDS micelles, which normally interfere with peptideseparations, to completely separate from the peptides, allowing distinct peptide bands to resolve.

5.2 Criterion Tris-Tricine/Peptide Gel Composition


5.3 Criterion Tris-Tricine/Peptide Gel Selection Guide

Criterion Tris-Tricine/peptide gels are available in either a single percentage gel or a linear gradient gel.Peptide Gel Optimal Separation16.5% 1.5–30 kD10–20% 1–40 kD

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5.4 Tris-Tricine/Peptide Buffers

Running buffer, 1x 100 mM Tris100 mM Tricine0.1% SDSDO NOT ADJUST pH

Sample buffer 200 mM Tris-HCl, pH 6.82% SDS40% glycerol0.04% Coomassie Blue G-2502% 2-mercaptoethanol or 350 mM DTT (added fresh)


Determine the appropriate protein concentration of your sample based on the detection method and loadvolume used. (See section 10.2 for approximate stain sensitivities.) Dilute 1 part sample with 2 parts samplebuffer and heat at 95ºC for 5 min.



Run time 120 min

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Section 6 Isoelectric Focusing

6.1 Introduction

Criterion IEF gels are cast with Bio-Rad’s Bio-Lyte® ampholytes, amphoteric molecules that set up a pHgradient across the gels. Proteins migrate in IEF gels to their neutral isoelectric point (pI), where the proteinhas zero net charge. Criterion IEF gels contain no denaturing agents, so all focusing is performed undernative conditions.

6.2 Criterion IEF Gel Composition

Gel buffer 2% ampholyte, pH 3–10, 5–8Cross-linker 3.0% CStacking gel NoneStorage buffer di H2O

Shelf life ~24 weeks, individual expiration date is printed on each cassette

6.3 Criterion IEF Gel Selection Guide

Criterion IEF gels are available in narrow and broad pH ranges.IEF gel pH Range

5–8 5–83–10 4–8.5

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6.4 IEF Buffers

Running buffers 1x Cathode Buffer20 mM lysine (free base)20 mM arginine (free base)

1x Anode Buffer7% phosphoric acid

Sample buffer 50% glycerol


Determine the appropriate protein concentration of your sample based on the detection method and loadvolume used. (See section 10.3 for approximate stain sensitivities.) Dilute 1 part sample with 2 parts samplebuffer.


Power conditions Stepwise:100 V constant 60 min250 V constant 60 min500 V constant 30 minStarting current: 5–25 mA/gelFinal current: 5–25 mA/gel

Run time 150 min

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Section 7 Protease Analysis by Zymogram PAGE

7.1 Introduction

Criterion zymogram gels are used to test for proteolytic activity when performing protein characterizations.Gels are cast with gelatin or casein, which act as substrates for proteases that are separated on the gel.Proteases are detected by renaturing the enzyme followed by a development period in which the proteasebreaks down the substrate. Zymogram gels are stained with Coomassie Blue R-250, which stains thesubstrate while leaving clear areas around active proteases.

7.2 Criterion Zymogram Composition

Gel buffer 0.375 M Tris-HCl, pH 8.6Cross-linker 2.6% CStacking gel 4% T, 2.6% CStorage buffer 0.375 M Tris-HCl, pH 8.6, 0.2% NaN3Shelf life ~8 weeks; individual expiration date is printed on each cassette

7.3 Criterion Zymogram Selection Guide

Criterion zymogram gels are available with either gelatin or casein as substrate and should be selected basedon substrate and separation range.Zymogram Gel Optimal Separation

10% zymogram gel with gelatin 30–150 kD12.5% zymogram gel with casein 20–120 kD

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7.4 Zymogram Buffers

Running buffer, 1x 25 mM Tris192 mM glycine0.1% SDSDO NOT ADJUST pH

Sample buffer 62.5 mM Tris-HCl, pH 6.84% SDS25% glycerol0.01% Coomassie Blue G-250


Determine the appropriate protein concentration of your sample based on the detection method and loadvolume used. (See section 10.4 for approximate stain sensitivities.) Dilute 1 part sample with 2 parts samplebuffer.



Run time 90 min

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Section 8Nondenaturing Nucleic Acid PAGE8.1 Introduction

Criterion TBE gels are ideal for separating small dsDNA fragments, especially PCR products. The uniformnature of DNA molecules provides samples with near-uniform charge to mass ratio, allowing nondenaturingnucleic acid PAGE to separate dsDNA by mass using a continuous TBE buffer system.

8.2 Criterion TBE Composition

Gel buffer 89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.3Cross-linker 3.3% CStacking gel 4% T, 3.3% CStorage buffer 89 mM Tris, 89 mM boric acid, 2 mM EDTAShelf life ~12 weeks; individual expiration date is printed on each cassette

8.3 Criterion TBE Gel Selection Guide

Criterion TBE gels are available in a selection of single percentages and gradients for the separation ofdsDNA.

Tris-HCl Gels Optimal Separation Tris-HCl Gradient Gels Optimal Separation5% 200–2,000 bp 4–20% 10–2,000 bp10% 50–1,500 bp15% 20–1,000 bp

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8.4 Nondenaturing Nucleic Acid PAGE Buffers

Running buffer, 1x 89 mM Tris89 mM boric acid2 mM EDTADO NOT ADJUST pH

Sample buffer, 5x 50 mM Tris-HCl, pH 8.05 mM EDTA25% glycerol0.2% Bromophenol Blue0.2% Xylene Cyanole FF


Determine the desired DNA concentration of your sample based on the detection method used. (See section10.5 for approximate stain sensitivities.) Dilute 4 parts sample with 1 part sample buffer (see section 8.4).



Run time 90 min

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Section 9 Denaturing Nucleic Acid PAGE9.1 Introduction

Criterion TBE-urea gels are ideal for separating small ssDNA and RNA fragments. Applications includeoligonucleotide analysis, RNase protection assays, and northern blotting.

9.2 Criterion TBE-Urea Gel Composition

Gel buffer 89 mM Tris, 89 mM boric acid, 2 mM EDTA, 7 M urea, pH 8.3Cross-linker 3.3% CStacking gel 4% T, 3.3% CStorage buffer 89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.3Shelf life ~8 weeks; individual expiration date is printed on each cassette

9.3 Criterion TBE-Urea Gel Selection Guide

Criterion TBE-urea gels are available in a range of single percentage gels.TBE-Urea Optimal Separation

5% 50–1,000 bases10% 25–300 bases15% 10–50 bases

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9.4 TBE-Urea Buffers

Running buffer, 1x 89 mM Tris89 mM boric acid2 mM EDTA

Sample buffer 89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.012% Ficoll0.01% Bromophenol Blue0.02% Xylene Cyanole FF7 M urea


Determine the desired ssDNA or RNA concentration for your sample based on the detection method used.(See section 10.6 for appropriate stain sensitivities.) Dilute 4 parts sample with 1 part sample buffer. (Seesection 9.4.)



Run time 90 min

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Section 10 Detection10.1 SDS-PAGE and Native PAGE Detection

Total Protein Gel StainsMethod Sensitivity Optimal Protein Load Advantages Disadvantages

Coomassie Blue R-250 36–47 ng ~0.5 µg/band Laboratory standard Requires MeOH

Bio-Safe™ Coomassie 8–24 ng ~0.5 µg/band Nonhazardous, uses More steps than stain no MeOH Coomassie R-250

Copper stain 6–12 ng ~0.2 µg/band Fast, reversible stain Negative stain, must be photographed;SDS-PAGE only

Zinc stain 6–12 ng ~0.2 µg/band High-contrast, fast, Negative stain, must reversible stain be photographed;

SDS-PAGE only

Silver Stain Plus™ kit 0.6–1.2 ng ~0.01 µg/band Simple, robust, mass Will not stainspectrometry compatible glycoproteins

Silver stain 0.6–1.2 ng ~0.01 µg/band Stains complex Not massproteins, i.e., glycoproteins spectrometryand lipoproteins compatible

SYPRO Orange protein 4–10 ng ~0.2 µg/band Will not stain nucleic Optimization requiredstain acids; mass for maximum

spectrometry compatible sensitivity

SYPRO Ruby protein 1–10 ng ~0.2 µg/band Broad dynamic range, Requires imaginggel stain simple robust protocol instrument for

maximum sensitivity

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Total Protein Gel StainsMethod Sensitivity Optimal Protein Load Advantages Disadvantages

SYPRO Ruby protein 2–8 ng ~0.2 µg/band Compatible with mass Multiple step protocol;blot stain spectrometry, Edman-based Requires imaging

sequencing, and standard instrument for immunological procedures maximum sensitivity

Colloidal gold stain 1 ng ~0.1 µg/band Sensitive, one step Not compatible withnylon membranes

Enhanced colloidal 10–100 pg ~0.1 µg/band Increases sensitivity of Multiple stepsgold detection kit colloidal gold stain

Amido Black 100–1,000 ng ~5 µg/band Standard membrane Low sensitivitystain, economical

Immunoblot DetectionMethod Sensitivity Optimal Protein Load Advantages Disadvantages

4CN colorimetric (HRP) 500 pg ~0.25 µg/band Fast detection Results may fade

DAB colorimetric (HRP) 500 pg ~0.25 µg/band Fast detection Contains toxicchemicals

Opti-4CN colorimetric 100 pg ~0.05 µg/band Color does not fade More expensive than(HRP) 4CN

Amplified Opti-4CN™ 10 pg ~0.005 µg/band High sensitivity, low Amplification requirescolorimetric (HRP) background additional steps

BCIP/NBT colorimetric 100 pg ~0.05 µg/band Sensitive; multiple antigens May detect endogenous (AP) enzyme activity

Amplified alkaline 10 pg ~0.005 µg/band High sensitivity Amplification requires phosphatase additional steps

Immun-Star™ 10 pg ~0.005 µg/band Long-lasting signal, Requires visualizationchemiluminescent (AP) short and multiple on film or instrumentation

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10.2 Peptide Gel Staining

Peptides and small proteins are prone to diffusion and loss during staining. The following protocol uses afixing step to prevent sample loss and is suitable for detection of bands as low as 10–20 ng.

Fixative Solution40% methanol10% acetic acid

Coomassie Brilliant Blue G-250 Stain Solution0.025% Coomassie Blue G-25010% acetic acid

Destain Solution10% acetic acid

Place gels in fixative solution and equilibrate for 30 min. Stain gels with Coomassie Brilliant Blue G-250 stainsolution for 1 hr. Stain should only be used once. Reuse of stain could result in lose of sensitivity. Destain gels3 times for 15 min or until the desired background is achieved. Some peptides may not be completely fixedand may diffuse out of the gels if fixing and staining times are greatly exceeded.

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10.3 IEF Gel Staining

Samples on IEF gels can be detected using multiple methods. Use the following table as a guide to select anappropriate staining method.

Method Sensitivity Optimal Protein Load Advantages Disadvantages

IEF stain 36–47 ng ~0.5 µg/band Simple, no fixation required Requires MeOH

Silver Stain Plus kit 0.6–1.2 ng ~0.01 µg/band Simple, robust, mass Requires TCAspectrometry compatible fixation

Silver stain 0.6–1.2 ng ~0.01 µg/band Stains complex Requires TCA proteins, i.e. glycoproteins fixationand lipoproteins

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10.4 Zymogram Gel Staining

Prior to staining zymogram gels, sample proteases must be first be renatured and allowed to break down thesubstrate contained in the gel. The following protocol provides basic guidelines for detection. Optimal resultsshould be determined empirically.

Renaturing Solution2.5% Triton X-100

Development Solution50 mM Tris200 mM NaCl5 mM CaCl2 (anhydrous)0.02% Brij-35Adjust to pH 7.5

Staining Solution40% methanol10% acetic acid0.5% Coomassie Blue R-250

Destaining Solution40% methanol10% acetic acid

Place gels in renaturing solution for 30 min at room temperature. Incubate gels in development solution at 37ºC for a minimum of 4 hr. Highest sensitivity is typically achieved with overnight incubations. Optimal resultsshould be determined empirically. Stain gels with Coomassie Brilliant Blue R-250 staining solution for at least1 hr at room temperature. Destain until clear bands appear against the blue background, approximately30–60 min.

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10.5 TBE Gel Staining

Use the following table as a guide to select an appropriate staining method.


Ethidium bromide 10 ng ~0.10 µg/band Classic fluorescent DNA stain Carcinogenic

Silver stain 1.0–2.0 ng ~0.5 µg/band More sensitive than Multiple stepsethidium bromide

10.6 TBE-Urea Gel StainingSample on denaturing nucleic acid gels can be detected using multiple methods. Use the following table asa guide to select an appropriate staining method.


Ethidium bromide 10 ng ~0.10 µg/band Classic fluorescent DNA stain Carcinogenic

Radiant® Red 10 ng ~0.10 µg/band Fast single-step protocol RNA and ssDNA only

Silver stain 1.0–2.0 ng ~0.5 µg/band More sensitive than Multiple stepsethidium bromide

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Section 11 TroubleshootingImproper storage of Criterion gels can produce numerous artifacts. Criterion gels should be stored flat at 4ºC.Avoid freezing or prolonged storage above 4ºC. If you suspect your gels have been stored improperly, DONOT USE THEM.

Problem Possible Cause Solution

Samples do not migrate into gel Tape at the bottom of the cassette Remove tapenot removed

Insufficient buffer in integral buffer chamber Fill integral buffer chamber with 60 ml running buffer

Insufficient lower electrode buffer Fill both halves of the lower buffer tank with400 ml running buffer when running two gels

Electrical disconnection Check electrodes and connections

Bands “smile” across gel, band pattern Excess heating of gel Check buffer composition curves upward at both sides of the gel Completely fill both halves of the lower buffer

tank with 400 ml running buffer when runningtwo gels

Do not exceed recommended runningconditions

Skewed or distorted bands, lateral Excess salt in samples Remove salts from sample by dialysis orband spreading desalting column prior to sample

preparation

Insufficient sample buffer Check buffer composition and dilution or wrong formulation instructions

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Problem Possible Cause Solution

Vertical streaking Overloaded samples Dilute sample

Selectively remove predominant protein inthe sample

Sample precipitation Centrifuge samples to remove particulatesprior to sample loading

Gels run too fast, provide poor resolution, Running buffer is too concentrated Check buffer compositionand gel temperature is too high

Artifact bands at ~60–70 kD Possible skin keratin contamination Clean all dishware and wear gloves while handling and loading gel

Filter all solutions through nitrocellulose

Use 10% iodoacetamide to eliminate keratin bands

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Section 12 Ordering Information12.1 Criterion Gels

12+2 Comb 18-Well Comb 26-Well Comb Prep+2 Comb IPG Comb IPG+1 CombCriterion Tris-HCl Gels 45 µl samples 30 µl samples 15 µl samples 800 µl samples 11 cm ReadyStrip 11 cm ReadyStrip5% Tris-HCl 345-0001 345-0002 345-0003 345-0004 - -7.5% Tris-HCl 345-0005 345-0006 345-0007 345-0008 - -10% Tris-HCl 345-0009 345-0010 345-0011 345-0012 345-0013 345-010112.5% Tris-HCl 345-0014 345-0015 345-0016 345-0017 345-0018 345-010215% Tris-HCl 345-0019 345-0020 345-0021 345-0022 - -18% Tris-HCl 345-0023 345-0024 345-0025 345-0026 - -4–15% Tris-HCl 345-0027 345-0028 345-0029 345-0030 345-0031 345-01034–20% Tris-HCl 345-0032 345-0033 345-0034 345-0035 345-0036 345-01048–16% Tris-HCl 345-0037 345-0038 345-0039 345-0040 345-0041 345-010510.5–14% Tris-HCl 345-9949 345-9950 345-9951 345-9952 345-9953 345-010610-20% Tris-HCl 345-0042 345-0043 345-0044 345-0045 345-0046 345-0107

12+2 Comb 18-Well Comb 26-Well Comb Prep+2 CombCriterion TBE Gels 45 µl samples 30 µl samples 15 µl samples 800 µl samples5% TBE 345-0047 345-0048 345-0049 345-005010% TBE 345-0051 345-0052 345-0053 345-005415% TBE 345-0055 345-0056 345-0057 345-00584–20% TBE 345-0059 345-0060 345-0061 345-0062

12+2 Comb 18-Well Comb 26-Well Comb Prep+2 CombCriterion Peptide Gels 45 µl samples 30 µl samples 15 µl samples 800 µl samples16.5% Peptide 345-0063 345-0064 345-0065 345-006610–20% Peptide 345-0067 345-0068 345-0069 345-0070

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12+2 Comb 18-Well Comb 26-Well Comb Prep+2 CombCriterion IEF Gels 45 µl samples 30 µl samples 15 µl samples 800 µl samplesIEF pH 3–10 345-0071 345-0072 345-0073 345-0074IEF pH 5–8 345-0075 345-0076 345-0077 345-0078

12+2 Comb 18-Well Comb 26-Well Comb Prep+2 CombCriterion Zymogram Gels 45 µl samples 30 µl samples 15 µl samples 800 µl samples10% Zymogram, gelatin 345-0079 345-0080 345-0081 -12.5% Zymogram, casein 345-0082 345-0083 345-0084 -

12+2 Comb 18-Well Comb 26-Well Comb Prep+2 CombCriterion TBE-Urea Gels 45 µl samples 30 µl samples 15 µl samples 800 µl samples5% TBE-Urea 345-0085 345-0086 345-0087 -10% TBE-Urea 345-0088 345-0089 345-0090 -15% TBE-Urea 345-0091 345-0092 345-0093 -

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12.2 Criterion Gel Accessories

345-9901 Criterion Empty Cassettes, 1.0 mm with 12+2 comb, 10345-9902 Criterion Empty Cassettes, 1.0 mm with 18-well comb, 10345-9903 Criterion Empty Cassettes, 1.0 mm with 26-well comb, 10345-9904 Criterion Empty Cassettes, 1.0 mm with prep+2 comb, 10345-9905 Criterion Empty Cassettes, 1.0 mm with IPG comb, 10

165-6006 Criterion Sample Loading Guide, 12+2 well, 1165-6007 Criterion Sample Loading Guide, 18-well, 1165-6008 Criterion Sample Loading Guide, 26-well, 1

12.3 Buffers

Premixed Running Buffers Premixed Sample Buffers161-0732 10x Tris/Glycine/SDS, 1 L 161-0737 Laemmli Sample Buffer, 30 ml*161-0772 10x Tris/Glycine/SDS, 5 L 161-0738 Native Sample Buffer, 30 ml161-0734 10x Tris/Glycine, 1 L 161-0739 Tricine Sample Buffer, 30 ml161-0771 10x Tris/Glycine, 5 L 161-0763 IEF Sample Buffer, 30 ml161-0744 10x Tris/Tricine/SDS, 1 L 161-0764 Zymogram Sample Buffer, 30 ml161-0761 10x IEF Anode Buffer, 250 ml 161-0767 Nucleic Acid Sample Buffer, 5x, 10 ml161-0762 10x IEF Cathode Buffer, 250 ml 161-0768 TBE-Urea Sample Buffer, 30 ml161-0733 10x Tris/Boric Acid/EDTA, 1 L161-0770 10x Tris/Boric Acid/EDTA, 5 L161-0765 Zymogram Renaturation Buffer, 125 ml * Requires addition of 2-mercaptoethanol or DTT

161-0766 Zymogram Development Buffer, 125 ml

Individual Reagents161-0719 Tris, 1 kg161-0718 Glycine, 1 kg161-0301 SDS, 100 g161-0710 2-Mercaptoethanol, 25 ml161-0611 Dithiothreitol, 5 g161-0404 Bromophenol Blue, 10 g

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12.4 Detection Reagents

Total Protein Gel Stains Total Protein Blot Stains161-0436 Coomassie Blue R-250 Stain Solution, 1 L 170-3127 SYPRO Ruby Protein Blot Stain, 200 ml161-0438 Coomassie Blue R-250 Destain Solution, 1 L 170-6527 Colloidal Gold Total Protein Stain, 500 ml161-0400 Coomassie Brilliant Blue R-250, 10 g 170-6517 Enhanced Colloidal Gold Detection Kit161-0786 Bio-Safe Coomassie Stain, 1 L 161-0402 Amido Black 10B, 25 g

161-0470 Copper Stain and Destain Kit161-0440 Zinc Stain and Destain Kit

161-0449 Silver Stain Plus Kit161-0443 Bio-Rad Silver Stain Kit

170-3120 SYPRO Orange Protein Stain, 500 µl170-3125 SYPRO Ruby Protein Gel Stain, 1 L161-0434 IEF Gel Staining Solution, 1 L

Immunoblot Detection170-6431 HRP Conjugate Substrate Kit, 4CN170-6535 HRP Color Development Reagent, DAB170-8238 Amplified Opti-4CN Kit170-8235 Opti-4CN Substrate Kit170-6432 BCIP/NBT AP Conjugate Substrate Kit170-6412 Amplified Alkaline Phosphatase Kit170-5012 Immun-Star Substrate Pack

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12.5 Blotting Membranes

162-0232 0.2 µm Nitrocellulose/Filter Paper Sandwich, 8.5 x 13.5 cm, 20 pack162-0233 0.2 µm Nitrocellulose/Filter Paper Sandwich, 8.5 x 13.5 cm, 50 pack162-0234 0.45 µm Nitrocellulose/Filter Paper Sandwich, 8.5 x 13.5 cm, 20 pack162-0235 0.45 µm Nitrocellulose/Filter Paper Sandwich, 8.5 x 13.5 cm, 50 pack162-0236 Sequi-Blot™ PVDF/Filter Paper Sandwich, 8.5 x 13.5 cm, 20 pack162-0237 Sequi-Blot PVDF/Filter Paper Sandwich, 8.5 x 13.5 cm, 50 pack

12.6 Protein and DNA Standards

161-0362 Precision Protein™ Unstained Standards (10–250 kD), 1,500 µl, 150 applications161-0372 Precision Protein Prestained Standards (10–250 kD), 500 µl, 50 applications161-0324 Kaleidoscope™ Prestained Standards, 500 µl, 50 applications161-0326 Polypeptide SDS-PAGE Standards (1.4–26.6 kD), 200 µl, 400 applications161-0310 IEF Standards, pI range 4.45–9.6, 250 µl, 500 applications

170-8351 20 bp EZ Load™ Molecular Ruler (20–1,000 bp), 50 µg, 100 applications170-8352 100 bp EZ Load Molecular Ruler (100–1,000 bp), 25 µg, 100 applications170-8353 100 bp PCR EZ Load Molecular Ruler (100–3,000 bp), 40 µg, 100 applications170-8200 AmpliSize® Molecular Ruler (50–2,000 bp), 25 µg, 50 applications

12.7 Equipment

165-6001 Criterion Cell, includes tank, lid with power cables, three sample loading guides170-4070 Criterion Blotter with Plate Electrodes170-4071 Criterion Blotter with Wire Electrodes

Brij is a trademark of ICI Americas, Inc. Coomassie is a trademark of Imperial Chemical Industries PLC. Ficoll is a trademark of AmershamPharmacia Biotech. SYPRO is a trademark of Molecular Probes, Inc. Bio-Rad is licensed to sell SYPRO products for research use only,under US patent 5,616,502. Triton is a trademark of Union Carbide.

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