Criterion Precast Gels ™ Instruction Manual and Application Guide
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Contents Chapter 1: Criterion™ Precast Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.2 Gel Formulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1.
Chapter 4: Native PAGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 4.2 Criterion Gel Selection Guide for Native PAGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 4.2.
Chapter 8: Protease Analysis by Zymogram PAGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 8.2 Criterion Zymogram Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 8.2.1 Gel Composition . . . . . . . . . . . . . . . . .
Chapter 12: Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 12.1 SDS-PAGE and Native PAGE Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 12.2 Peptide Gel Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 12.3 IEF Gel Staining . . . .
1 Criterion™ Precast Gels 1.1 Introduction Criterion precast gels are an effective system for performing polyacrylamide gel electrophoresis (PAGE). These 13.3 x 8.7 cm gels are wider and longer than traditional mini format gels, and their innovative, easy-to-use design produces excellent resolution while accommodating more samples per gel.
Criterion Precast Gels 1.2 Gel Formulations Criterion precast gels are available in a range of formulations for virtually every electrophoresis application (Table 1.1). All Criterion gels are composed of polyacrylamide with a bisacrylamide crosslinker, and they are available in a selection of single percentages and gradients. Table 1.1. Criterion precast gel formulations.
Instruction Manual and Application Guide 1.5 Storage Conditions Table 1.2. Storage conditions for Criterion precast gels. Store gels flat. Shelf life is from date of manufacture; expiration dates are printed on the cassettes.
2 Setup and Basic Operation 2.
Instruction Manual and Application Guide 2.2 Required Materials n Criterion™ precast gels n Criterion or Criterion™ Dodeca™cell n PowerPac™ Basic or PowerPac HC power supply (or equivalent) n Sample buffer n Running buffer (460 ml per gel) 2.3 Setting Up and Running Criterion Gels in the Criterion Cell 1. Each Criterion gel is packaged in a plastic storage tray. Remove the cover of the tray by lifting the corner tab and pulling it diagonally across the package. Remove the gel from the package.
Criterion Precast Gels 4. Press down firmly to break the seals on both sides of the cassette. The cassette splits open approximately ¹/3 of the way. Alternatively, open the gel cassette by sliding the tapered back of the comb into the slits on either side of the cassette. C 5. Pull the two halves of the cassette apart from the top to completely expose the gel (C). 6.
3 SDS-PAGE 3.1 Introduction Criterion™ precast gels provide versatile systems for the separation of proteins by either molecular weight (SDS-PAGE) or mass-to-charge ratio (native PAGE). (See Chapter 4 for native PAGE applications and protocols.) This versatility is possible because Criterion gels are made without SDS, allowing the sample buffer and running buffer to determine the separation mechanism. SDS-PAGE relies on a discontinuous buffer system.
Criterion Precast Gels Table 3.1. Criterion precast gels for SDS-PAGE.
Instruction Manual and Application Guide 3.2.2 Criterion Tris-HCl and Criterion Stain Free Gels These Tris-HCl, Laemmli gels use discontinuous glycinate and chloride ion fronts to form moving boundaries to stack and then separate denatured proteins by size. They are run using standard Laemmli sample buffer and Tris/glycine/SDS running buffer.
Criterion Precast Gels Migration charts for protein standards on Criterion Tris-HCl, Criterion TGX, and TGX Stain-Free gels. 3.2.3 Criterion XT Bis-Tris Gels Criterion XT Bis-Tris gels are based on a Bis-Tris HCl buffer system (pH 6.4) that uses discontinuous chloride and MES or MOPS ion fronts to form moving boundaries that stack and separate denatured proteins by size. This chemistry of XT Bis-Tris gels allows maximum stability and consistent results with a shelf life of at least 12 months.
Instruction Manual and Application Guide Gel Percentage Optimum Separation Range XT MES Buffer XT MOPS Buffer 10% 2.5–200 kD 14–220 kD 12% 1–30 kD 6–66 kD 2.5–200 kD 10–300 kD 4–12% Migration charts for protein standards on Criterion XT Bis-Tris gels. 3.2.4 Criterion XT Tris-Acetate Gels Criterion XT Tris-acetate gels are based on a Tris-acetate buffer system (pH 7.0).
Criterion Precast Gels 3.3 SDS-PAGE Buffers Table 3.2. Recommended Criterion precast gels and buffers for SDS-PAGE.
Instruction Manual and Application Guide 3.5 Running Conditions Run conditions and times are approximate. Conditions may vary depending on water and buffer conductivity, which vary from one lab setting to the next. Multiply current by the number of gels run. Table 3.3. Running conditions for SDS-PAGE with Criterion gels in the Criterion cell. Do not run different gel formulations at the same time.
4 Native PAGE 4.1 Introduction In native PAGE, proteins are prepared in nonreducing, nondenaturing sample buffer, which maintains native structure and mass-to-charge ratios. Separation is also performed in the absence of SDS and reducing agents. Though native PAGE uses the same moving boundary described for SDS-PAGE (see Section 3.1), protein mobility depends on a number of factors besides molecular weight, including the shape and charge of the protein.
Instruction Manual and Application Guide Gel Composition Crosslinker 2.6% C Stacking gel 4% T, 2.6% C Shelf life ~12 months at 2–8°C; expiration date is printed on each cassette 4.2.2 Criterion Tris-HCl and Criterion Stain Free Gels These Tris-HCl Laemmli gels are run using native sample buffer and Tris/glycine running buffer.
Criterion Precast Gels 4.4 Sample Preparation In the absence of SDS, the net charge of a polypeptide is determined by its amino acid composition and the pH of the sample buffer. Only polypeptides with a net negative charge migrate into Criterion gels under native conditions. Most polypeptides have an acidic or slightly basic pI (~3–8). These proteins can be separated using the following standard protocol: 1.
Criterion Stain Free™ System 5 5.1 Introduction The Criterion Stain Free system, which comprises the Gel Doc™ EZ imager, Image Lab™ software, and Criterion™ TGX Stain-Free™ and Criterion Stain Free precast gels, eliminates the time-consuming staining and destaining steps required by other protein detection methods.
Criterion Precast Gels 5.2 Criterion Stain Free Workflow Perform Electrophoresis SDS-PAGE (Chapter 3) Native PAGE (Chapter 4) 2-D Electrophoresis (Chapter 11) Activate/Image Gels (Chapter 5) Stain the Gels for Total Protein Blot the Gels (Chapter 12) (Chapter 13) Analyze the Separation 5.3 Electrophoresis with Criterion TGX Stain-Free Gels Criterion TGX Stain-Free gels (and Criterion Stain Free gels) are made and packaged without SDS, so they can be used for both SDS and native PAGE applications.
6 Peptide Analysis 6.1 Introduction Criterion™ Tris-Tricine/peptide gels are optimized for separating peptides and proteins with molecular weight <10,000. Peptide-SDS complexes move more slowly through these gels, allowing the faster SDS micelles that normally interfere with peptide separations to separate completely from peptides. This enables resolution of distinct peptide bands. 6.2 Criterion Tris-Tricine/Peptide Gels 6.2.1 Gel Composition Gel buffer 1.0 M Tris-HCl, pH 8.45 Crosslinker 2.
Criterion Precast Gels 6.4 Sample Preparation 1. Determine the appropriate concentration of sample to load (depends on the load volume and the detection method used; see Chapter 12 for approximate stain sensitivities). 2. Dilute the sample with at least an equivalent volume of sample buffer (catalog #161-0739) and reducing agent (β-mercaptoethanol, for example). Heat the diluted sample at 90–95°C for 5 min, or at 70°C for 10 min. For example, combine: 5 μl sample 4.
Isoelectric Focusing (IEF) 7 7.1 Introduction Isoelectric focusing (IEF) separates proteins by their net charge rather than molecular weight. Criterion™ IEF gels are cast with Bio-Rad’s Bio-Lyte® ampholytes, amphoteric molecules that generate a pH gradient across the gels. Proteins migrate to their isoelectric point (pI), the pH at which the protein has no net charge. Criterion IEF gels contain no denaturing agents, so IEF is performed under native conditions. 7.2 Criterion IEF Gels 7.2.
Criterion Precast Gels 7.4 Sample Preparation 1. Determine the appropriate concentration of sample to load (depends on the load volume and the detection method used). 2. Dilute the sample with at least an equivalent volume of sample buffer. For example, combine: 5 μl sample 5 μl sample buffer 10 μl total volume 7.
8 Protease Analysis by Zymogram PAGE 8.1 Introduction Criterion™ zymogram gels are used to test for proteolytic activity. Gels are cast with gelatin or casein, which acts as a substrate for proteases that are separated in the gel under nonreducing conditions. Proteases are detected by first renaturing the enzymes and then allowing them to break down the substrate.
Criterion Precast Gels 8.4 Sample Preparation 1. Determine the appropriate protein concentration of your sample based on the detection method and load volume used. (See Chapter 12 for approximate stain sensitivities.) 2. Dilute 1 part sample with 1 part sample buffer. Do not heat the samples. 8.5 Running Conditions 24 Power conditions 125 V constant Starting current 90–120 mA/gel Final current 35–55 mA/gel Run time 90 min Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.
Nondenaturing Nucleic Acid PAGE 9 9.1 Introduction Criterion™ TBE gels are used to separate small double-stranded DNA (dsDNA) fragments, particularly PCR products. DNA molecules have nearly uniform mass-to-charge ratios, allowing nondenaturing nucleic acid PAGE to separate dsDNA by mass using a continuous TBE buffer system. 9.2 Criterion TBE Gels 9.2.1 Gel Composition Gel buffer 89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.3 Crosslinker 3.3% C Stacking gel 4% T, 3.
Criterion Precast Gels 9.4 Sample Preparation Determine the DNA concentration of your sample based on the detection method used. (See Chapter 12 for approximate stain sensitivities.) Dilute 4 parts sample with 1 part sample buffer. 9.5 Running Conditions Table 9.1. Running conditions for nondenaturing nucleic acid PAGE with Criterion gels in the Criterion cell.
10 Denaturing Nucleic Acid PAGE 10.1 Introduction Criterion™ TBE-urea gels are used for separation of small RNA and single-stranded DNA (ssDNA) fragments. Applications include oligonucleotide analysis, RNase protection assays, and northern blotting. 10.2 Criterion TBE-Urea Gels 10.2.1 Gel Composition Gel buffer 89 mM Tris, 89 mM boric acid, 2 mM EDTA, 7 M urea, pH 8.3 Crosslinker 3.3% C Stacking gel 4% T, 3.3% C Storage buffer 89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.
Criterion Precast Gels 10.5 Running Conditions Table 10.1. Running conditions for denaturing nucleic acid PAGE with Criterion gels in the Criterion cell. 5% Gels 10% Gels 15% Gels Power conditions 200 V constant 200 V constant 200 V constant Initial 40–45 mA 20–33 mA 18–22 mA Final 20–25 mA 14–18 mA 10–15 mA 90 min 90 min 90 min Expected current (per gel) Run time 28 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.
11 2-D Electrophoresis 11.1 Introduction Criterion™ precast gels are available for second-dimension PAGE in 2-D electrophoresis workflows. The IPG-well gels accommodate 11 cm IPG strips. Criterion™ Any kD™ gels are particularly well suited for 2-D electrophoresis applications. The transition from first- to second-dimension gel electrophoresis involves: n Equilibration of the resolved IPG strips in a reducing buffer containing SDS n Placing the IPG strip on top of the second-dimension gel 11.
12 Detection 12.1 SDS-PAGE and Native PAGE Detection Following electrophoresis, either stain the gel or use the Criterion Stain Free™ system to visualize proteins in the gel. n n Refer to Table 12.1 for a comparison of total protein stains For Criterion™ TGX Stain-Free™ and Criterion Stain Free™ gels, immediately place the gel on the tray of the Gel Doc™ EZ imager; no additional fixation or rinsing steps are required. If desired, stain with any compatible stains (Table 12.1) following imaging.
Instruction Manual and Application Guide Sensitivity (Lower Limit) Stain SYPRO Ruby protein gel stain Flamingo™ fluorescent gel stain Optimum Protein Load (µg/Band) 1–10 ng 0.25–0.5 ng Advantages Disadvantages Imaging ~0.1 Broad dynamic range ~0.
Criterion Precast Gels 12.4 Zymogram Gel Staining Prior to staining zymogram gels, sample proteases must first be renatured and allowed to break down the substrate contained in the gel. The following protocol provides basic guidelines for detection. Optimal results should be determined empirically. Renaturing solution 2.5% Triton X-100 Development solution 50 mM Tris, 200 mM NaCl, 5 mM CaCl2 (anhydrous), 0.02% Brij-35 Adjust to pH 7.5 Staining solution 40% methanol, 10% acetic acid, 0.
13 Blotting 13.1 Introduction Western blotting is an electrophoretic technique used to move proteins from a gel onto a solid support such as a nitrocellulose or PVDF membrane. The membrane can be used for immunological or biochemical analyses or demonstration of protein-protein or protein-ligand interactions. Below are guidelines for western blotting of Criterion™ precast gels onto nitrocellulose or PVDF membranes using either wet or semi-dry transfer techniques.
Criterion Precast Gels b) Open the cassette and place it in the back (large) compartment of the tray so the red plate with the handle is vertical (anode) and the black plate (cathode) is horizontal and submerged in transfer buffer. c) Assemble the sandwich as shown, placing the gel closest to the black side of the cassette and the membrane closest to the red plate. Use a blot roller to remove air trapped between the layers of the blot assembly.
Instruction Manual and Application Guide Cassette top (–) electrode (cathode) Top ion reservoir stack Gel Membrane Bottom ion reservoir stack Cassette bottom (+) electrode (anode) Assembly of the gel blot sandwich with the Trans-Blot Turbo system. Table 13.1. Placement of cassettes in the Trans-Blot Turbo cell.
Criterion Precast Gels 13.2.4 Semi-Dry Transfer Using the Trans-Blot® SD Cell 1. Equilibrate the gels and membranes (for example, in transfer buffer; see Appendix B for buffer recipes) for 20 min prior to blot assembly. 2. Assemble the blot for transfer using the Trans-Blot SD semi-dry transfer system. 3. Connect the Trans-Blot SD cell to a PowerPac HC power supply and begin transfer at 10–15 V.
Instruction Manual and Application Guide To visualize total protein on blots using the Gel Doc EZ imager, refer to Section 5.4. 13.4 Immunodetection After transfer, blots are ready for downstream processing. While all protein and antibody combinations are different and may require optimization, a general protocol for the immunodetection of a large number of protein and antibody combinations is provided (see Appendix B for buffer formulations). 1.
14 Troubleshooting Table 14.1. Troubleshooting electrophoresis and detection with Criterion™ gels. For more troubleshooting tips, refer to the Criterion cell, Criterion blotter, and Trans-Blot® SD cell instruction manuals, or contact Technical Support.
Instruction Manual and Application Guide Problem Artifactual bands at 60–70 kD Cause Skin keratin contamination Solution Clean all dishware, and wear gloves while handling and loading gels Filter all solutions (0.2–0.
A Quick Start Guide The following instructions are for electrophoresis of Criterion™ precast gels using the Criterion system. Prepare Buffers Running buffer (1x) Dilute 100 ml 10x stock (catalog #161-0732) with 900 ml diH2O. Sample buffer Use Laemmli sample buffer (catalog #161-0737). Prepare Gels and Assemble Electrophoresis Cell Remove the comb and tape from the gels and assemble the electrophoresis cell. Fill the inner and outer buffer chambers with running buffer.
Table A.1. Running conditions for SDS-PAGE in the Criterion cell. Do not run different gel formulations at the same time.
B Buffers Running Buffers 42 10x SDS-PAGE (1 L) (catalog #161-0732) 250 mM Tris, 1.92 M glycine, 1% SDS, pH 8.3 10x Native PAGE (1 L) (catalog #161-0734) 250 mM Tris, 1.92 M glycine, pH 8.3 10x Tris-Tricine (1 L) (catalog #161-0744) 1 M Tris, 1 M Tricine, 1% SDS, pH 8.3 10x TBE (1 L) (catalog #161-0741) 890 mM Tris, 890 mM boric acid, 20 mM EDTA Tris base Glycine SDS diH2O Do not adjust the pH (~pH 8.
Instruction Manual and Application Guide Sample Buffers 2x SDS-PAGE (Laemmli, 30 ml) 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 25% glycerol, 0.01% (catalog #161-0737) bromophenol blue, 5% β-mercaptoethanol (added fresh) 0.5 M Tris-HCl, pH 6.8 50% Glycerol 1.0% Bromophenol blue 10% SDS diH2O 3.75 ml 15.0 ml 0.3 ml 6.0 ml to 30 ml Add β-mercaptoethanol (50 µl to 950 µl sample buffer) before use. 2x Native PAGE (30 ml) 62.5 mM Tris-HCl, pH 6.8, 40% glycerol, 0.
Criterion Precast Gels TBE-urea (30 ml) (catalog #161-0768) Store at 4°C 89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.0, 12% Ficoll, 0.01% bromophenol blue, 0.02% xylene cyanole, 7 M urea Tris base Boric acid EDTA Ficoll Bromophenol blue Xylene cyanole FF Urea diH2O 0.32 g 0.165 g 17.5 mg 3.6 g 3 mg 6 mg 12.6 g to 30 ml 0.5 M Tris-HCl, pH 6.8 (1 L) (catalog #161-0799) Store at 4°C Tris base diH2O Adjust to pH 6.8 with HCl diH2O 60.6 g ~900 ml 10% SDS (250 ml) (catalog #161-0416) SDS diH2O 1.
Instruction Manual and Application Guide C Related Literature Bulletin # Title 4006183 Criterion™ Cell Instruction Manual 4006197 Criterion™ Dodeca™ Cell Instruction Manual 10014472 Gel Doc™ EZ System Installation Guide 10019634 Stain-Free Sample Tray Instruction Manual 4006190 Criterion Blotter Instruction Manual 4006066 Trans-Blot® SD Semi-Dry Transfer Cell Quick Reference Guide 10020688 Trans-Blot® Turbo™ Blotting System Instruction Manual 1703940 Trans-Blot SD Semi-Dry T
Criterion Precast Gels D Ordering Information Criterion TGX™ Gels 12+2 Well (45 µl/well) 18-Well (30 µl/well) 26-Well (15 µl/well) IPG+1 Well (11 cm IPG Strip) Prep+2 Well (800 µl/well) 7.
Instruction Manual and Application Guide Criterion™ Tris-HCl Gels 12+2 Well (45 µl/well) 18-Well (30 µl/well) 26-Well (15 µl/well) IPG+1 Well (11 cm IPG Strip) Prep+2 Well (800 µl/well) 5% 345-0001 345-0002 345-0003 — — 7.5% 345-0005 345-0006 345-0007 — 345-0008 10% 345-0009 345-0010 345-0011 345-0101 345-0012 12.
Criterion Precast Gels Criterion Zymogram Gels 12+2 Well (45 µl/well) 18-Well (30 µl/well) 26-Well (15 µl/well) 10% Zymogram, gelatin 345-0079 345-0080 345-0081 12.
Instruction Manual and Application Guide Catalog # Description Protein Standards 161-0363 Precision Plus Protein™ Unstained Standards (10–250 kD), 1 ml, 100 applications 161-0373 Precision Plus Protein All Blue Prestained Standards (10–250 kD), 500 μl, 50 applications 161-0374 Precision Plus Protein Dual Color Standards (10–250 kD), 500 μl, 50 applications 161-0375 Precision Plus Protein™ Kaleidoscope™ Standards (10–250 kD), 500 μl, 50 applications 161-0376 Precision Plus Protein™ WesternC™ Sta
Criterion Precast Gels Catalog # Description Premixed Sample Buffers 161-0737 Laemmli Sample Buffer, 30 ml1 161-0738 Native Sample Buffer, 30 ml 161-0791 XT Sample Buffer, 4x, 10 ml 161-0792 XT Reducing Agent, 20x, 1 ml 161-0739 Tricine Sample Buffer, 30 ml 161-0763 IEF Sample Buffer, 30 ml 161-0764 Zymogram Sample Buffer, 30 ml 161-0767 Nucleic Acid Sample Buffer, 5x, 10 ml 161-0768 TBE-Urea Sample Buffer, 30 ml Component Reagents 161-0719 Tris, 1 kg 161-0718 Glycine, 1 kg 161-0301 SDS,
Instruction Manual and Application Guide Catalog # Description Immunoblot Detection Reagents 161-0385 Precision Plus Protein™ WesternC™ Pack 170-5070 Immun-Star™ WesternC™ Chemiluminescent Kit, 100 ml 170-6431 HRP Conjugate Substrate Kit, 4CN 170-6535 HRP Color Development Reagent, DAB, 5 g 170-8238 Amplified Opti-4CN™ Substrate Kit 170-8235 Opti-4CN Substrate Kit 170-6432 AP Conjugate Substrate Kit 170-5012 Immun-Star™ Substrate Pack Blotting Membranes 162-0232 0.
Criterion Precast Gels 52 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.
Instruction Manual and Application Guide Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.
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