Bio-Rad Mini-PROTEAN TGX Precast Gels for 2-D Electrophoresis User Manual

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Mini-PROTEAN® Precast Gels

Instruction Manual and Application Guide

Page 2

Bio-Rad Technical Support

For help and technical advice, please contact the Bio-Rad Technical Support department. In the United States, the Technical Support department is open Monday–Friday, 5:00 am–5:00 pm, Pacific Time.

Phone: 1-800-424-6723

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Contents

Chapter 1: Mini-PROTEAN® Precast Gels ............................................. 1

1.1 Introduction ................................................................... 1

1.2 Gel Formulations ............................................................... 2

1.3 Comb Configurations ........................................................... 2

1.4 Specifications ................................................................. 2

1.5 Storage Conditions ............................................................ 3

1.6 Important Notes ............................................................... 3

Chapter 2: Setup and Basic Operation ............................................... 4

2.1 Workflow Overview ............................................................. 4

2.2 Required Materials ............................................................. 5

2.3 Setting Up and Running Mini-PROTEAN Gels in the Mini-PROTEAN® Tetra Cell............... 5

2.4 Removing the Gel .............................................................. 7

Chapter 3: SDS-PAGE............................................................. 8

3.1 Introduction ................................................................... 8

3.2 Mini-PROTEAN® TGX™ and Mini-PROTEAN® TGX Stain-Free™ Gels ....................... 8

3.3 SDS-PAGE Buffers ............................................................ 10

3.4 Sample Preparation............................................................ 10

3.5 Running Conditions ............................................................ 10

Chapter 4: Native PAGE .......................................................... 12

4.1 Introduction .................................................................. 12

4.2 Mini-PROTEAN TGX and Mini-PROTEAN TGX Stain-Free Gels........................... 12

4.3 Native PAGE Buffers .......................................................... 13

4.4 Sample Preparation............................................................ 13

4.5 Running Conditions ............................................................ 13

Chapter 5: Stain-Free System ..................................................... 14

5.1 Introduction .................................................................. 14

5.2 Stain-Free Workflow ........................................................... 15

5.3 Electrophoresis with Mini-PROTEAN TGX Stain-Free Gels .............................. 15

5.4 Stain-Free Detection ........................................................... 15

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Chapter 6: Peptide Analysis ....................................................... 16

6.1 Introduction .................................................................. 16

6.2 Mini-PROTEAN Tris-Tricine Gels ................................................. 16

6.2.1 Gel Composition .......................................................... 16

6.2.2 Gel Selection Guide........................................................ 16

6.3 Peptide Analysis Buffers ........................................................ 17

6.4 Sample Preparation............................................................ 17

6.5 Running Conditions ............................................................ 17

Chapter 7: Nondenaturing Nucleic Acid PAGE ........................................ 18

7.1 Introduction .................................................................. 18

7.2 Mini-PROTEAN TBE Gels ....................................................... 18

7.2.1 Gel Composition .......................................................... 18

7.2.2 Gel Selection Guide........................................................ 18

7.3 Nondenaturing Nucleic Acid PAGE Buffers .......................................... 19

7.4 Sample Preparation............................................................ 19

7.5 Running Conditions ............................................................ 19

Chapter 8: Denaturing Nucleic Acid PAGE ........................................... 20

8.1 Introduction .................................................................. 20

8.2 Mini-PROTEAN TBE-Urea Gels ................................................... 20

8.2.1 Gel Composition .......................................................... 20

8.2.2 Gel Selection Guide........................................................ 20

8.3 Denaturing Nucleic Acid PAGE Buffers ............................................. 21

8.4 Sample Preparation............................................................ 21

8.5 Running Conditions ............................................................ 21

Chapter 9: 2-D Electrophoresis .................................................... 22

9.1 Introduction .................................................................. 22

9.2 Equilibration.................................................................. 22

9.3 Agarose Overlay .............................................................. 22

9.4 Second-Dimension Electrophoresis................................................ 22

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Chapter 10: Detection............................................................ 23

10.1 SDS-PAGE and Native PAGE Detection ........................................... 23

10.2 Peptide Gel Staining .......................................................... 24

10.3 TBE Gel Staining ............................................................ 24

10.4 TBE-Urea Gel Staining ....................................................... 24

Chapter 11: Blotting ............................................................. 25

11.1 Introduction ................................................................. 25

11.2 Transfer ................................................................... 25

11.2.1 Transfer Buffers .......................................................... 25

11.2.2 Wet Transfer Using the Mini Trans-Blot® Module................................. 25

11.2.3 Transfer Using the Trans-Blot® Turbo™ System.................................. 26

11.2.4 Semi-Dry Transfer Using the Trans-Blot® SD Cell ................................ 27

11.3 Total Protein Blot Stains ....................................................... 28

11.4 Immunodetection ............................................................ 28

Chapter 12: Troubleshooting ...................................................... 29

Appendix A: Quick Start Guides.................................................... 31

SDS-PAGE (Mini-PROTEAN TGX Gels) ............................................. 32

Native PAGE (Mini-PROTEAN TGX Gels) ............................................ 33

Peptide Analysis (Mini-PROTEAN Tris-Tricine Gels) .................................... 34

Nondenaturing Nucleic Acid PAGE (Mini-PROTEAN TBE Gels)............................ 35

Denaturing Nucleic Acid PAGE (Mini-PROTEAN TBE-Urea Gels) .......................... 36

Appendix B: Buffers ............................................................. 37

Appendix C: Related Literature .................................................... 40

Appendix D: Ordering Information .................................................. 41

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Mini-PROTEAN

Precast Gels

1.1 Introduction

Mini-PROTEAN precast gels are 7.2 cm x 8.6 cm gels designed for performing polyacrylamide gel electrophoresis (PAGE) with the Mini-PROTEAN family of vertical electrophoresis cells, which includes the Mini-PROTEAN® Tetra and Mini-PROTEAN® 3 Dodeca™ cells and the discontinued Mini-PROTEAN II and Mini-PROTEAN 3 cells. The Mini Trans-Blot®, Trans-Blot® Turb o™, and Trans-Blot® SD blotting cells and precut membrane sandwiches are also available for blotting applications with these gels.

Features of Mini-PROTEAN precast gels include:

Outlined and numbered well that simplify sample loading and identiﬁcation

Capacity for up to 15 samples per gel

Bottom-open cassette design for easy gel handling and blotting setup

Easy-to-open cassette for faster downstream processing

Reference line at the bottom of the cassette indicates where the run should stop

(for optimum resolution across the separation range)

Excellent staining quality and transfer efﬁciency

No gel foot to remove prior to blotting

Mini-PROTEAN® TGX Stain-Free™ formulations, which enable rapid 5 min gel imaging

without staining and destaining

Comb (available in a range of options)

Numbered well outlines

Reference line for monitoring progress of the run

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Mini-PROTEAN Precast Gels

1.2 Gel Formulations

Mini-PROTEAN precast gels are composed of polyacrylamide with a bisacrylamide crosslinker, and they are available in a range of formulations (Table 1.1) and in a selection of single percentages and gradients.

Table 1.1. Mini-PROTEAN precast gel formulations.

Application Gel Formulation Sample Buffer Running Buffer

SDS-PAGE Mini-PROTEAN TGX™ Laemmli Tris/glycine/SDS Mini-PROTEAN TGX Stain-Free

Native PAGE Mini-PROTEAN TGX Native Tris/glycine Mini-PROTEAN TGX Stain-Free

Peptide analysis Mini-PROTEAN Tris-Tricine Tricine Tris/Tricine/SDS

dsDNA separation Mini-PROTEAN TBE Nucleic acid Tris/boric acid/EDTA (TBE)

ssDNA and RNA Mini-PROTEAN TBE-urea TBE-urea TBE separation

1.3 Comb Configurations

Comb Type Well Volume

10-well 50 μl

10-well 30 μl

12-well 20 μl

15-we ll 15 μl

8 + 1 well* 30 μl

IPG/prep 7 cm ReadyStrip™ IPG strip (450 μl)

1.4 Specifications

Gel material Polyacrylamide

Gel dimensions 7.2 x 8.6 cm

Gel thickness 1.0 mm

Resolving gel height 6.2 cm (5.6 cm for 50 μl well)

Cassette dimensions 8.5 x 10 cm

Cassette material Styrene copolymer

Comb material Polycarbonate

Running buffer 750 ml for 1–2 gels, 1,000 ml for 3–4 gels (Mini-PROTEAN Tetra cell) 325 ml for 1–2 gels (Mini-PROTEAN II or Mini-PROTEAN 3 cell)

Multichannel pipet compatible.

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Instruction Manual and Application Guide

1.5 Storage Conditions

Table 1.2. Storage conditions for Mini-PROTEAN precast gels. Store gels flat. Shelf life is from date of manufacture;

expiration dates are printed on the packaging.

Storage Temperature Gel Formulation Shelf Life

2–8°C Mini-PROTEAN TGX 12 months

Mini-PROTEAN TGX Stain-Free 12 months

Mini-PROTEAN Tris-Tricine 12 weeks

Mini-PROTEAN TBE 12 weeks

Mini-PROTEAN TBE-urea 8 weeks

1.6 Important Notes

Use each Mini-PROTEAN precast gel as soon as possible after removing it from the storage pouch.

Improper storage of Mini-PROTEAN precast gels can produce artifacts. Store gels ﬂat and at 2–8°C. Avoid freezing or prolonged storage above 8°C. If your gels have been stored improperly, discard them.

Do not run more than one gel type in the same apparatus at the same time. Different gel percentages and formulations have different conductivities and different run times.

With the Mini-PROTEAN Tetra cell:

When running 1–2 gels:

Use the electrode assembly (with banana plugs), not the companion running module

(without banana plugs)

Do not place the companion running module in the tank. Doing so generates

excessive heat and degrades the quality of the electrophoretic separation

When running 3–4 gels, use both the electrode assembly and companion running module

When using voltages >200 V, ﬁll the outer buffer chamber to the 4 gel (800 ml) mark

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Setup and Basic

Operation

2.1 Workflow Overview

Prepare sample and running buffers

Assemble Electrophoresis Cell

Prepare and Load Samples

Prepare Buffers

Prepare Gels and

Dilute in sample buffer

Perform Electrophoresis

SDS-PAGE (Chapter 3)

Native PAGE (Chapter 4)

Peptide Analysis (Chapter 6)

Nondenaturing Nucleic Acid PAGE (Chapter 7)

Denaturing Nucleic Acid PAGE (Chapter 8)

2-D Electrophoresis (Chapter 9)

Analyze the Separation

Blot the Gels (Optional)

(Chapter 10)

(Chapter 11)

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Instruction Manual and Application Guide

2.2 Required Materials

Mini-PROTEAN® precast gels

Mini-PROTEAN® Tetra cell (or Mini-PROTEAN® 3 Dodeca™, Mini-PROTEAN II or

Mini-PROTEAN 3 cell)

PowerPac™ Basic or PowerPac HC power supply (or equivalent); PowerPac HV or

PowerPac Universal required for high-voltage applications (>300 V)

Sample buffer

Running buffer (750 ml for 1–2 gels; 1,000 ml for 3–4 gels or when running at

voltages >200 V)

Opening lever (catalog #456-0000)

2.3 Setting Up and Running Mini-PROTEAN Gels in the Mini-PROTEAN Tetra Cell

1. Remove the gels from the storage pouch and prepare them for assembly:

a. Remove the comb: Position thumb on the indentation (middle of comb) and remove the comb

by pulling upward in one smooth motion.

b. Remove the tape: Pull gently to remove the green tape from the bottom of the cassette.

If necessary, use the opening key or comb to help remove the tape at the corners.

c. Rinse the wells: Use a syringe, wash bottle, or disposable transfer pipet to rinse the wells with

Remove the comb

Remove the tape

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Mini-PROTEAN Precast Gels

running buffer. Straighten the sides of the wells, if necessary.

2. Set the electrode assembly to the open

position on a clean, ﬂat surface (A).

3. Place the gel cassettes into the electrode assembly. Two cassettes are required to create a functioning assembly; when using 1 or 3 gels, use the buffer dam (included with the cell) to complete the assembly.

a. Place the ﬁrst cassette with the short plate

facing inward and so the gel rests at a 30° angle away from the center of the electrode assembly. Make sure the electrode assembly remains balanced and does not tip over.

b. Place the second gel or buffer dam on the

other side of the electrode assembly, again by resting the gel on the supports. The gels rest at 30° angles, one on either side of the electrode assembly, tilting away from the center of the frame (B).

4. Gently push both gels toward each other, making sure that they rest ﬁrmly and squarely against the green gasket that is built into the electrode assembly. Align the short plates to ensure the edge sits just below the notch at the top of the green gasket (C).

5. While gently squeezing the gel cassettes (or cassette and buffer dam) against the green gaskets (maintaining constant pressure and with both gels in place), slide the green arms of the clamping frame one at a time over the gels, locking them into place (D,E).

Clamping frame

Gasket

Notch

Gel cassette

Short plate

Long plate

Gel support

6. The wing clamps of the electrode assembly lift each gel cassette up against the notch in the green gasket, forming a seal. Check again that

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Instruction Manual and Application Guide

the short plates sit just below the notch at the top of the green gasket (C).

If running more than 2 gels, repeat steps 2–6 with the

companion running module.

7. Place the electrophoresis module into the tank (F) and

ﬁll the buffer chambers with 1x running buffer:

200 ml in the inner buffer chamber

550 ml (1–2 gels) or 800 ml (3–4 gels, or

>200 V) in the outer buffer chamber

8. Wash the sample wells with running buffer (if this was not done earlier).

9. Load samples and run the gels using the running

conditions appropriate to your application. Stop the run when the dye front reaches the reference line imprinted on the bottoms of the cassettes.

2.4 Removing the Gel

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

2. Remove the lid from the tank and remove the gels from the cell. Pour off and discard the running buf fer.

3. To open the cassette, align the arrow on the opening lever with the arrows marked on the cassette and insert the lever between the cassette plates at indicated locations. Apply downward pressure to break each seal. Do not twist the lever.

4. Pull the two plates apart from the top of the cassette, and gently remove the gel.

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SDS-PAGE

3.1 Introduction

Mini-PROTEAN® TGX™ (Tris-Glycine eXtended shelf life) gels provide a versatile system for separating 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 the gels are made without SDS; this allows the sample buffer and running buffer to determine the separation mechanism.

SDS-PAGE relies on a discontinuous buffer system. Two ions differing in electrophoretic mobility (glycinate and chloride) form a moving boundary when voltage is applied. Proteins have an intermediate mobility that causes them to concentrate, or stack, into a narrow zone at the beginning of electrophoresis. As that zone moves through the gel, the sieving effect of the polyacrylamide gel matrix causes proteins of different molecular weighs to move at different rates. This stacking effect is responsible for the high resolving power of SDS-PAGE: the sample is loaded in a relatively broad zone, and the moving boundary concentrates the proteins into sharp bands prior to separation.

Protein samples for SDS-PAGE are prepared using SDS and a thiol reducing agent, usually β-mercaptoethanol or dithiothreitol (DTT). SDS forms complexes with proteins, giving them a rodlike shape and similar mass-to-charge ratio. The reducing agent disrupts disulﬁde bonds between and within proteins, allowing complete denaturation and dissociation. Heat treatment in the presence of SDS and reducing agent effectively eliminates the effects of native charge and higher order structure on electrophoretic mobility, so the migration distance depends primarily on molecular weight.

Molecular weight is estimated by plotting the logarithm of protein molecular weight vs. the relative mobility (Rf) of the protein (Rf = distance migrated by the protein/distance migrated by the dye front) or by using the point-to-point semilog interpolation method in Quantity One® or Image Lab™ software. Refer to bulletins 3133, 3144, and 10014472 for more information.

3.2 Mini-PROTEAN TGX and

Mini-PROTEAN® TGX Stain-Free™ Gels

Mini-PROTEAN TGX gels are Laemmli-like gels that have a proprietary modiﬁcation that extends shelf life to 12 months and enhances separation characteristics relative to conventional gel types. They are run using standard Laemmli sample buffer and Tris/glycine/SDS running buffer, and they generate protein migration patterns that are similar to those observed with standard Laemmli Tris-HCl gels.

Two types of TGX formulations are available:

Mini-PROTEAN TGX — Laemmli-like, extended shelf life gels

Mini-PROTEAN TGX Stain-Free — Laemmli-like, extended shelf life gels with trihalo

compounds that allow rapid ﬂuorescent detection of proteins with the stain-free system, eliminating staining and destaining steps for faster results (see Chapter 5 for more details)

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Mini-PROTEAN® TGX™ Precast Gels

Instruction Manual and Application Guide

Both gel types gels are available in polyacrylamide single percentages and gradients. Use the protein migration charts and tables to select the gel type that optimizes resolution of your sample:

Use single-percentage gels to separate bands of similar molecular weight. Optimum separation

occurs in the lower half of the gel, so use a percentage in which the protein migrates to the lower half of the gel

Use gradient gels to separate samples containing a broad range of molecular weights. Gradient

gels allow resolution of both high- and low-molecular weight bands on the same gel. Larger pore sizes at the top of the gel permit resolution of larger molecules, smaller pore sizes toward the bottom of the gel restrict excessive separation of small molecules

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 package

Gel Percentage Optimum Separation Gel Percentage Optimum Separation Range Range

7.5% 40–200 kD 4–15% 20–250 kD

10% 30–150 kD 4–20% 10–200 kD

12% 20–120 kD Any kD™ 10 –100 kD

Broad Range Unstained

200

116

97.4

21.5

200

116

97.4

21.5

14.4

™

Any kD

4–20%4–15%12%10%7.5%

200

116

97.4

21.5

14.4

6.5

200

116

97.4

21.5

14.4

6.5

200

116

97.4

21.5

14.4

6.5

250

150

100

Precision Plus Protein™ Unstained

™

Any kD

4–20%4–15%12%10%7.5%

250

150

100

250

150

100

250

150

100

250

150

100

250 150

100

200

116

97.4

Migration charts for protein standards on Mini-PROTEAN TGX and Mini-PROTEAN TGX Stain-Free gels.

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Mini-PROTEAN Precast Gels

3.3 SDS-PAGE Buffers

See Appendix B for buffer formulations. Do not adjust pH.

Running buffer (1x) 25 mM Tris, 192 mM glycine, 0.1% SDS

Dilute 100 ml 10x stock (catalog #161-0732) with 900 ml deionized water (diH2O).

Sample buffer (2x) 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 25% (v/v) glycerol, 0.01% bromophenol blue, 5% β-mercaptoethanol or 100 mM DTT (added fresh)

Use Laemmli sample buffer (catalog #161-0737) and add β-mercaptoethanol or DTT before use.

Sample buffer (4x) 250 mM Tris-HCl, pH 6.8, 4% LDS, 40% (w/v) glycerol, 0.02% bromophenol blue, 15% beta-mercaptoethanol or 200 mM DTT (added fresh)

Use 4x Laemmli sample buffer (catalog #161-0747) and add β-mercaptoethanol or DTT before use.

3.4 Sample Preparation

1. Determine the appropriate concentration of sample to load (depends on the load volume and the detection method used; see Chapter 10 for approximate stain sensitivities).

2. Dilute the sample with sample buffer with added reducing agent.

2x: dilute 1 part sample with 1 part sample buffer.

4x: dilute 3 parts sample with 1 part sample buffer.

For nonreducing conditions, omit the reducing agent.

3. Heat the diluted sample at 90–95°C for 5 min or at 70°C for 10 min.

3.5 Running Conditions

Run conditions and times are approximate. Run times represent the time required for the dye front to reach the line at the bottom of the cassette. 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 3.1. Standard running conditions for SDS-PAGE in the Mini-PROTE AN Tetra cell.

Gel Optimum Range Run Conditions Run Time

7.5% 40–200 kD

10% 30–150 kD 300 V constant: 12% 20–120 kD Starting current (per gel): 55 –75 mA 15–20 min 4–15% 20–250 kD Final current (per gel): 45–70 mA (Fill outer buffer volume 4–20% 10–200 kD to the 4-gel mark) Any kD 10–100 kD

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Instruction Manual and Application Guide

Table 3.2. Alternative running conditions for SDS-PAGE in the Mini-PROTEAN Tetra cell.

100 V 200 V

Run time 85–95 min 30– 40 min

Expected current (per gel)

Initial 15 –20 mA 25–50 mA

Final 5–10 mA 20–31 mA

Expected temperature 25°C 25–35°C

Outer buf fer volume

1–2 Gels 2-gel mark 2-gel mark 3–4 Gels 4-gel mark 4-gel mark

Table 3.3. PowerPac power supply recommendations.

# Gels 100 V 200 V 300 V

1–2 Basic/HC/HV/Universal Basic/HC/HV/Universal Basic/HV/Universal

3–4 Basic/HC/HV/Universal Basic/HC/HV/Universal HV/Universal

4–8 HC/HV/Universal HC/HV/Universal Universal

9–10 HC/Universal HC/Universal Universal

11–12 HC/Universal HC/Universal Universal

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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 other than molecular weight, including the shape and charge of the protein. Protein-protein interactions may be retained during native PAGE, so some proteins may separate as multisubunit complexes. Consequently, native PAGE is not suitable for molecular weight determination.

The nonreducing and nondenaturing environment of native PAGE allows protein separation with retention of biological activity. Because native structure is retained, native PAGE can enable separation of proteins with the same molecular weight.

4.2 Mini-PROTEAN® TGX™ and

Mini-PROTEAN® TGX Stain-Free™ Gels

Mini-PROTEAN TGX gels are Laemmli-like gels that have a proprietary modiﬁcation that extends their shelf life to 12 months and enhances separation characteristics relative to conventional gel types. They are run using standard native sample buffer and Tris/glycine running buffer, and they generate protein migration patterns that are similar to those observed with standard Laemmli Tris-HCl gels.

Two types of TGX formulations are available:

Mini-PROTEAN TGX — Laemmli-like, extended shelf life gels

Mini-PROTEAN TGX Stain-Free — Laemmli-like, extended shelf life gels with trihalo

compounds that allow rapid ﬂuorescent detection of proteins with the stain-free system, eliminating staining and destaining steps for faster results (see Chapter 5 for more details)

These gels are available in a selection of polyacrylamide single percentages and gradients, and because they contain no SDS, they can be used for either SDS- or native PAGE applications.

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 the packaging

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Instruction Manual and Application Guide

4.3 Native PAGE Buffers

See Appendix B for buffer formulations. Do not adjust pH.

Running buffer (1x) 25 mM Tris, 192 mM glycine Dilute 100 ml 10x stock (catalog #161-0734) with 900 ml diH2O.

Sample buffer 62.5 mM Tris-HCl, pH 6.8, 40% (w/v) glycerol, 0.01% (w/v) bromophenol blue (catalog # 161-0738)

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 gel during electrophoresis, which is a function of the sample buffer, gel buffer, and running buffer. Only polypeptides with a net negative charge migrate into 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. Determine the desired protein concentration and load volume of your sample based on the detection method used (see Chapter 10 for approximate stain sensitivities).

2. Dilute the sample with an equal volume of native sample buffer (do not heat the samples).

For example, combine: 5 μl sample

5 μl native sample buffer (catalog #161-0738)

10 μl total volume

Strongly basic proteins (pl >8.5) have a net positive charge and will not enter a Mini-PROTEAN TGX gel under native conditions using Tris/glycine buffer. To allow polypeptides with a net positive charge to migrate into a native gel, change the polarity of the electrodes by reversing the color-coded jacks when connecting to the power supply.

4.5 Running Conditions

Running conditions for native PAGE are similar to the standard running conditions used for SDS-PAGE (Section 3.4). If elevated temperature is a concern, run native PAGE at lower voltage; at lower voltages, runs require more time to complete.

Table 4.1. Standard running conditions for native PAGE with one (1) gel in the Mini-PROTEAN Tetra cell. Run conditions and times are approximate and assume a constant voltage of 200 V. When running more than one gel, current will differ but temperature and run time should be close to those listed.

Current (mA) at 200 V Gel Initial Final Temperature Run Time

1 Gel (buffer to 2-gel mark)

7.5% 35–37 17–20 28–30°C 38 –40 min

10% 12%

4–15% 50–55 25–28 30–33°C 30–34 min 4–20% Any kD

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Stain-Free System

5.1 Introduction

Bio-Rad’s stain-free system eliminates the time-consuming staining and destaining steps required by other protein detection methods. Mini-PROTEAN® TGX Stain-Free™ gels include unique trihalo compounds that allow rapid ﬂuorescent detection of proteins with stain free-enabled imaging systems — without staining.

The trihalo compounds in the gels react with tryptophan residues in a UV-induced reaction to produce ﬂuorescence, which can be easily detected (by stain free-enabled imagers) within gels or on lowﬂuorescence PVDF membranes. Activation of the trihalo compounds in the gels adds 58 Da moieties to available tryptophan residues and is required for protein visualization. Proteins that do not contain tryptophan residues cannot be detected using this system. The sensitivity of the stain-free system is comparable to staining with Coomassie Brilliant Blue for proteins with a tryptophan content >1.5%; sensitivity superior to Coomassie staining is possible for proteins with a tryptophan content >3%.

Imaging systems that can be used with the stain-free system include the Gel Doc™ EZ and ChemiDoc™ MP systems (with Image Lab™ software).

Molecular weights of proteins can be estimated by a regression method using Image Lab software. The software generates a standard curve using the molecular weight and relative mobility (Rf) of standard proteins (Rf = distance migrated by the protein/distance migrated by the dye front). The standard curve is then used to estimate the molecular weights of sample proteins.

Beneﬁts of the stain-free system include:

Elimination of staining and destaining steps for faster results

Automated gel imaging and analysis

No background variability within a gel or between gels (as is often seen with standard

Coomassie staining)

Reduced organic waste by not requiring acetic acid and methanol for staining or destaining

Visualization of transferred (blotted) proteins on low ﬂuorescence PVDF membranes

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Page 21

Instruction Manual and Application Guide

5.2 Stain-Free Workflow

Perform Electrophoresis

SDS-PAGE (Chapter 3)

Native PAGE (Chapter 4)

2-D Electrophoresis (Chapter 9)

Activate/Image Gels

(Chapter 5)

Stain the Gels for Total Protein

(Chapter 10)

Blot the Gels

(Chapter 11)

Analyze the Separation

5.3 Electrophoresis with Mini-PROTEAN TGX Stain-Free Gels

Mini-PROTEAN TGX Stain-Free gels are made and packaged without SDS, so they can be used for both SDS and native PAGE applications. To perform electrophoresis with these gels, prepare the sample and running buffers, set up the Mini-PROTEAN Tetra cell, and perform the run as directed in Chapters 2–4.

Use unstained standards with Mini-PROTEAN TGX Stain-Free gels, as some prestained standards are not compatible with stain-free technology. To monitor electrophoresis, use 10 µl of a 1:1 mixture of Precision Plus Protein™ unstained (catalog #161-0363) and Precision Plus Protein All Blue protein standards (catalog #161-0373).

5.4 Stain-Free Detection

Image Mini-PROTEAN TGX Stain-Free gels and blots in a compatible imager. The imager activates the reaction between the proteins and trihalo compounds in the gel to enable visualization.

Immediately place the gel in the tray of the imager; no ﬁxation or rinsing steps are required.

Prolonged rinsing may diminish image quality and lead to gel deformation

If desired, stain the gel with any TGX-compatible stains after imaging. Certain stains, if used

prior to imaging, eliminate detection capability

Refer to the Gel Doc EZ Stain-Free Sample Tray Instruction Manual (bulletin 10019634) or the ChemiDoc MP System with Image Lab Software Instruction Manual (bulletin 10022469) for detailed instructions.

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Page 22

Peptide Analysis

6.1 Introduction

Mini-PROTEAN® Tris-Tricine peptide analysis 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, which normally interfere with peptide separations, to completely separate from peptides. This enables resolution of distinct peptide bands.

6.2 Mini-PROTEAN Tris-Tricine Gels

6.2.1 Gel Composition

Gel buffer 1.0 M Tris-HCl, pH 8.45

Crosslinker 2.6% C

Stacking gel 4% T, 2.6% C

Storage buffer 1.0 M Tris-HCl, pH 8.45, NaN

Shelf life 12 weeks at 2–8°C; expiration date is printed on the packaging

6.2.2 Gel Selection Guide

Gel Percentage Optimum Separation Range

16.5% 1.5–30 kD

10–20% 1–40 kD

Migration charts for protein standards on Mini-PROTEAN Tris-Tricine gels.

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Page 23

Instruction Manual and Application Guide

6.3 Peptide Analysis Buffers

See Appendix B for buffer formulations. Do not adjust pH unless instructed to do so.

Running buffer (1x) 100 mM Tris, 100 mM Tricine, 0.1% SDS Dilute 100 ml 10x stock (catalog #161-0744) with 900 ml diH2O

Sample buffer 200 mM Tris-HCl, pH 6.8, 2% SDS, 40% glycerol, 0.04% Coomassie (catalog #161-0739) Brilliant Blue G-250, 2% β-mercaptoethanol or 100 mM DTT (added fresh)

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 10 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

4.75 μl Tricine sample buffer (catalog #161-0739)

0.25 μl β-mercaptoethanol (catalog #161-0710)

10 μl total volume

6.5 Running Conditions

Table 6.1. Running conditions for one (1) Mini-PROTEAN Tricine gel in the Mini-PROTEAN Tetra cell. Run conditions and times are approximate and assume a constant voltage of 100 V. When running more than one gel, current will differ.

16.5% Gels 10–20% Gels

Power conditions 100 V constant 100 V constant

Expected current (per gel)

Initial 65 mA 65 mA

Final 35 mA 35 mA

Run time 100 min 100 min

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Page 24

Nondenaturing Nucleic

Acid PAGE

7.1 Introduction

Mini-PROTEAN® 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.

7.2 Mini-PROTEAN TBE Gels

7.2.1 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, 3.3% C

Storage buffer 89 mM Tris, 89 mM boric acid, 2 mM EDTA, NaN

Shelf life 12 weeks at 2–8°C; expiration date is printed on the packaging

7.2.2 Gel Selection Guide

Gel Percentage Optimum Separation Range

5% 200–2,000 bp

10% 50–1,500 bp

15% 20–1,000 bp

4–20% 10–2,000 bp

Migration charts for DNA standards on Mini-PROTEAN TBE gels.

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Page 25

Instruction Manual and Application Guide

7.3 Nondenaturing Nucleic Acid PAGE Buffers

See Appendix B for buffer formulations. Do not adjust pH unless directed to do so.

Running buffer (1x) 89 mM Tris, 89 mM boric acid, 2 mM EDTA Dilute 100 ml 10x stock (catalog #161-0733) with 900 ml diH2O

Sample buffer (5x) 50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 25% (w/v) glycerol, 0.2% bromophenol (catalog #161-0767) blue, 0.2% xylene cyanole FF

7.4 Sample Preparation

Determine the DNA concentration of your sample based on the detection method used. (See Chapter 10 for approximate stain sensitivities.) Dilute 4 parts sample with 1 part sample buffer.

7.5 Running Conditions

Table 7.1. Running conditions for nondenaturing nucleic acid PAGE with one (1) Mini-PROTEAN TBE gel in the Mini-PROTEAN Tetra cell. Run conditions and times are approximate and assume a constant voltage of 100 V. When running

more than one gel, current will differ.

5% Gels 10% Gels 15% Gels 4–20% Gels

Power conditions 100 V constant 100 V constant 100 V constant 100 V constant

Expected current (per gel)

Initial 15 mA 15 mA 15 mA 15 mA

Final 10 mA 10 mA 10 mA 10 mA

Run time 45–60 min 60 –75 min 75–90 min 90–105 min

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Page 26

Denaturing Nucleic

Acid PAGE

8.1 Introduction

Mini-PROTEAN® 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.

8.2 Mini-PROTEAN TBE-Urea Gels

8.2.1 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 8.3, NaN

Shelf life 8 weeks at 2–8°C; expiration date is printed on the packaging

8.2.2 Gel Selection Guide

Gel Percentage Optimum Separation Range

10% 25–300 nt

15% 10–50 nt

Migration charts for DNA standards on Mini-PROTEAN TBE-urea gels.

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Page 27

Instruction Manual and Application Guide

8.3 Denaturing Nucleic Acid PAGE Buffers

See Appendix B for buffer formulations. Do not adjust pH unless directed to do so.

Running buffer (1x) 89 mM Tris, 89 mM boric acid, 2 mM EDTA Dilute 100 ml 10x stock (catalog #161-0733) with 900 ml diH2O

Sample buffer (5x) 89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.0, 12% Ficoll, (catalog #161-0768) 0.01% bromophenol blue, 0.02% xylene cyanole FF, 7 M urea

8.4 Sample Preparation

Determine the desired ssDNA or RNA concentration for your sample based on the detection method used. Dilute 4 parts sample with 1 part sample buffer.

8.5 Running Conditions

Table 8.1. Running conditions for denaturing nucleic acid PAGE with one (1) Mini-PROTEAN TBE-urea gel in the Mini-PROTEAN Tetra cell. Run conditions and times are approximate and assume a constant voltage of 200 V. When running

more than one gel, current will differ.

10% Gels 15% Gels

Power conditions 200 V constant 200 V constant

Expected current (per gel)

Initial 15 mA 15 mA

Final 10 mA 10 mA

Run time 45–60 min 60 –75 min

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Page 28

2-D Electrophoresis

9.1 Introduction

Mini-PROTEAN® precast gels are available for second-dimension PAGE in 2-D electrophoresis workﬂows. The IPG-well gels accommodate 7 cm IPG strips. Mini-PROTEAN® TGX Any kD™ gels are particularly well suited to 2-D electrophoresis applications.

The transition from ﬁrst-to second-dimension gel electrophoresis involves:

Equilibration of the resolved IPG strips in an SDS-containing, reducing buffer

Placing the IPG strip on top of the second-dimension gel (agarose overlay)

9.2 Equilibration

Equilibration ensures that proteins in the IPG strips are coated with SDS and that cysteines are reduced and alkylated. Use the equilibration protocols (bulletin 411009) and buffers in the ReadyPrep™ 2-D starter kit (catalog #163-2105), or other protocols and buffers used for Tris-HCl gels.

9.3 Agarose Overlay

Place the equilibrated IPG strip into the IPG well of the gel and overlay it with molten agarose to ensure good contact between the strip and gel.

1. Prepare 0.5% low-melt agarose (catalog #161-3111), 0.003% bromophenol blue (catalog #161-0404) in 1x Tris/glycine/SDS running buffer (or use ReadyPrep overlay agarose, catalog #163-2111).

2. Following equilibration, place the IPG strip, gel side up, on the back plate of the gel, above the IPG well. The “+” and pH range on the IPG strip should be on the left.

3. Using forceps, push the strip into the IPG well, taking care to not trap air bubbles under the strip. Push on the backing of the strip, not on the gel.

4. Using a disposable pipet, apply overlay agarose into the IPG well. Fill the well to the top of the inner plate. Dispense rapidly, as overlay agarose solidiﬁes quickly. To avoid bubbles, tilt the cassette slightly to allow bubbles to escape. Push gently on the plastic backings of the strip to free any trapped bubbles.

9.4 Second-Dimension Electrophoresis

Place the cassettes in to the Mini-PROTEAN® Tetra cell and start the run using the run conditions for SDS-PAGE. Use the migration of the bromophenol blue in the overlay agarose to monitor the progress of the run.

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Page 29

Detection

10.1 SDS-PAGE and Native PAGE Detection

Following electrophoresis, stain the gel with a total protein stain to visualize the proteins (Table 10.1).

Table 10.1. Total protein gel stains for use with Mini-PROTEAN gels.

Stain

Mini-PROTEAN TGX Gels

Coomassie R-250

™

Bio-Safe Coomassie

Zinc stain

Sensitivity

(Lower

Limit)

36 –47 ng ~0.5 Laboratory

8–28 ng ~0.5 Nonhazardous 43 070 51

6–12 ng ~0.2 High contrast, fast,

Optimum

Protein Load

(µg/Band)

Advantages

standard

reversible

Disadvantages

Requires methanol destaining

Negative SDSPAGE stain, must be photographed

Imaging

Photography with white light or transmission densitometry

Manual

Consult literature

4006082

Silver Stain Plus™ kit

Silver stain 0.6–1. 2 n g ~0.01 Stains complex

™

Dodeca silver stain kit

™

Oriole ﬂuorescent

gel stain

SYPRO Ruby protein gel stain

Flamingo ﬂuorescent gel stain

Stain-free imaging

Do not use zinc stain or Oriole ﬂuorescent gel stain to stain native gels.

™

0.6–1.2 n g ~0.01 Sensitive, robust, mass spectrometry compatible

proteins (glyco- or lipoproteins)

0. 5–1.2 n g ~ 0.1 Convenient staining for a large number of gels

~2 ng ~0.1 High sensitivity,

broad dynamic range, simple one-step protocol

1–10 ng ~ 0.1 Broad dynamic

range

0.25– 0.5 ng ~0.02 Broad dynamic range, mass spectrometry compatible

2–28 ng ~0.5 Rapid (<5 min),

compatible with blotting and mass spectrometry, simple protocol with no additional reagents

Does not stain glycoproteins well

Not mass spectrometry compatible

Requires laseror LED-based imaging instrument for maximum sensitivity

Requires tryptophan residues in proteins for detection

Fluorescence visualization with UV transillumination

Fluorescence visualization with UV, LED, or laser scanning

Fluorescence using stain- free compatible imaging system

LIT4 42

LIT34

4110 15 0

10017295

400 6173

10003321

10014472

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Page 30

Mini-PROTEAN Precast Gels

10.2 Peptide Gel Staining

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

Fixative solution 40% methanol, 10% acetic acid

Stain solution 0.025% (w/v) Coomassie Blue G-250, 10% acetic acid

Destain solution 10% acetic acid

Place gels in ﬁxative solution and equilibrate for 30 min. Stain gels with stain solution for 1 hr. Stain should be used only once; reuse may result in loss of sensitivity. Destain gels three times for 15 min or until the desired background is achieved. Some peptides may not be completely ﬁxed and may diffuse out of the gels if ﬁxing and staining times are greatly exceeded.

10.3 TBE Gel Staining

Use Table 10.2 as a guide to selecting an appropriate staining method.

Table 10.2. TBE gel detection methods.

Method

Ethidium bromide 50 ng Classic ﬂuorescent DNA stain Carcinogenic

Silver stain 1–2 ng More sensitive than ethidium bromide Requires multiple steps

SYBR® Green 0.02–2 ng High sensitivity Multiple steps, –20°C storage

SYBR® Safe 0.5 ng Non-hazardous Multiple steps

Sensitivity

(Lower Limit)

Advantages

Disadvantages

10.4 TBE-Urea Gel Staining

Use Table 10.3 as a guide to selecting an appropriate staining method.

Table 10.3. TBE-urea gel detection methods.

Method

Ethidium bromide 10 ng Classic ﬂuorescent DNA stain Carcinogenic

SYBR® Green 0.02–2 ng High sensitivity Requires multiple steps, −20°C

Silver stain 1–2 ng More sensitive than ethidium bromide Requires multiple steps

Sensitivity

(Lower Limit)

Advantages

Disadvantages

storage

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Page 31

Blotting

11.1 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 Mini-PROTEAN® precast gels onto nitrocellulose or PVDF membranes using either wet or semi-dry transfer techniques. After transfer, assess transfer efﬁciency using a total protein blot stain (see Section 11.3); with Mini-PROTEAN® TGX Stain-Free™ gels, transfer efﬁciency to low ﬂuorescence PVDF membranes may also be assessed using the Gel Doc™ EZ or ChemiDoc™ MP imager (see Chapter 5; activate the gel before blotting).

See Appendix B for buffer formulations. Do not adjust pH unless directed to do so.

11.2 Transfer

11.2.1 Transfer Buffers

Towbin buffer (1x) 25 mM Tris, 192 mM glycine, 20% (v/v) methanol (pH 8.3) Dilute 100 ml 10x stock (catalog #161-0734) with 400 ml diH2O. Add 200 ml methanol, then adjust volume to 1 L with diH2O.

Add SDS to 0.1% to promote transfer of high molecular weight proteins.

11.2.2 Wet Transfer Using the Mini Trans-Blot® Module

1. Equilibrate the gels in transfer buffer for 10–20 min prior to blot assembly.

2. Assemble the Mini Trans-Blot cassette. Place the gel closest to the black plate and the membrane closest to the red plate of the cassette. Use a roller to remove air trapped between the layers of the blot assembly.

Wet PVDF membranes in methanol before soaking in transfer buffer.

3. Place the assembled cassette into the transfer module and tank. The red cassette plate should face the red side of the transfer module. Repeat steps 2 and 3 for a second blot, if needed.

4. Add the cooling unit and stirbar, and ﬁll the tank with transfer buffer. Place the tank on a stir plate, and begin stirring to maintain even buffer temperature and ion concentration during the transfer.

5. Connect the Mini Trans-Blot cell to a suitable power supply and begin transfer.

For many proteins, excellent transfer efﬁciency is obtained in 30 min at a constant voltage of 100 V. For best results, optimize conditions for proteins of interest. Large proteins (>150 kD) may take 60 min, while smaller proteins (<30 kD) may transfer in 20 min. Refer to the Mini Trans-Blot Instruction Manual (bulletin

1703910) or the Protein Blotting Guide (bulletin 2895) for additional information.

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Page 32

Mini-PROTEAN Precast Gels

Foam pad

Filter paper Membrane

Gel Filter paper Foam pad

Assembly of the Mini-Trans-Blot cassette.

11.2.3 Transfer Using the Trans-Blot® Turbo™ System

1. Open the transfer pack and assemble the components on the cassette in the order shown. For best

results, use the roller to remove any air trapped between the layers. If using a single mini or midi sandwich, place the sandwich in the middle of the cassette bottom. With two mini gels, place the sandwiches on a midi stack with the foot of each gel facing the center of the stack.

2. Place the lid on the cassette and lock the lid in place by turning the knob clockwise, using the

symbols on the lid as a guide. Slide the cassette into the appropriate bay of the Trans-Blot Turbo cell. Each cassette and bay can hold up to two mini gels or one midi gel (Table 11.1).

3. Start the transfer. With the cassette in the cell, press TURBO and select the gel type. Press A:RUN

or B:RUN to begin the transfer. Press LIST to select a Bio-Rad optimized protocol (Table 11.2) or a user-deﬁned protocol. Press NEW to create and run a new protocol.

4. When transfer completes, RUN COMPLETE appears. Pull the cassette straight out of the slot and

unlock the lid. Disassemble the blotting sandwich.

Refer to the Trans-Blot Turbo Instruction Manual (bulletin 10020688) for complete instructions.

Cassette top (–) electrode (cathode)

Top ion reservoir stack

Gel

Membrane

Bottom ion reservoir stack

Assembly of the gel blot sandwich with the Trans-Blot Turbo system.

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Cassette bottom (+) electrode (anode)

Page 33

Instruction Manual and Application Guide

Table 11.1. Placement of cassettes in the Trans-Blot Turbo cell.

Acceptable Unacceptable

Option 1 Option 2 Option 1 Option 2

Upper bay (A) 1 mini gel 2 mini gels -or- 1 midi gel 1 mini gel 2 mini gels -or- 1 midi gel

-a nd/or- -a nd /or- -and- -and-

Lower bay (B) 1 mini gel 2 mini gels -or- 1 midi gel 2 mini gels -or- 1 midi gel 1 mini gel

Table 11.2. Trans-Blot Turbo transfer protocols.

Protocol Name MW, kD Time, Min 1 Mini Gel 2 Mini Gels or 1 Midi Gel

STA ND AR D SD Any 30 Up to 1.0 A, 25 V constant Up to 1.0 A, 25 V constant

1.5 MM GEL Any 10 2.5 A constant, up to 25 V 1.3 A constant, up to 25 V

HIGH MW >15 0 10 2.5 A constant, up to 25 V 1.3 A constant, up to 25 V

LOW MW <30 5 2.5 A constant, up to 25 V 1.3 A constant, up to 25 V

MIXED MW 5–15 0 7 2.5 A constant, up to 25 V 1.3 A constant, up to 25 V

1 Mini TGX 5–150 3 2.5 A constant, up to 25 V N/A

11.2.4 Semi-Dry Transfer Using the Trans-Blot® SD Cell

1. Equilibrate the gels in transfer buffer for 10–20 min.

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™ Basic power supply and begin transfer at 25 V.

Optimum transfer efﬁciency is generally obtained in 30 min; smaller proteins (<30 kD) may transfer more quickly, while proteins >150 kD may show increased transfer efﬁciencies at up to 60 min. Run times longer than 60 min are NOT recommended for semi-dry transfers. Refer to the Trans-Blot SD Instruction Manual (bulletin 1703940) or the Protein Blotting Guide (bulletin 2895) for additional information.

(–)

Filter paper

Gel

Membrane

Filter paper

(+)

Assembly of the Trans-Blot SD semi-dry cell.

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Page 34

Mini-PROTEAN Precast Gels

11.3 Total Protein Blot Stains

Total protein staining of a membrane provides an image of the complete protein pattern, which is required for the full characterization of speciﬁc antigens detected in complex protein mixtures. Gels shrink during staining, so comparison of an immunologically probed membrane to a stained gel is not practical. Instead, the exact location of a specifc antigen is determined by comparing two blotted membranes: one that has been probed with an antibody and the other stained for total protein.

Table 11.3. Total protein blot stains.

Method

SYPRO Ruby protein blot stain

Sensitivity

2–8 ng ~0.2 Compatible with

Protein

Load

(μg/Band)

Advantages

mass spectrometry, Edman-based sequencing, and standard immunological procedures

Disadvantages

Multistep protocol requires UV, LED, or laser imaging for maximum sensitivity

Imaging

Fluorescence visualization with UV, LED epi-illumination or laser scanning

Colloidal gold stain 1 ng ~0.1 High sensitivity;

single-step protocol

Anionic dyes (amido black, Coomassie R-250, Ponceau S, Fast Green FCF)

100–1,000 ng ~5.0 Inexpensive, rapid Low sensitivity

Incompatible with nylon membranes

Photography with epi-illumination or reﬂectance densitometry

To visualize total protein on blots using the stain-free system, see Section 5.4.

11.4 Immunodetection

After transfer, blots are ready for downstream processing. Though all protein and antibody combinations are different and may require optimization, a general protocol for immunodetection of a large number of protein and antibody combinations is listed below. See Appendix B for buffer formulations.

1. Immediately after transfer, place the membrane into Tris-buffered saline with Tween 20 (TTBS) containing blocking agent (for example, 3% BSA, 5% nonfat dry milk, 1% casein, or 1% gelatin) and incubate either for 1 hr at room temperature or overnight at 4°C.

2. Dilute the primary antibody in blocking solution (dilution is speciﬁed by the manufacturer). Incubate at room temperature with agitation for 1 hr.

3. Wash the blot with TTBS as directed (for example, ﬁve times, 5 min each at room temperature with agitation).

4. Dilute the secondary antibody into TTBS as speciﬁed by the manufacturer. Incubate the blot in the secondary solution at room temperature with agitation for 1 hr.

5. Wash the blot with TTBS ﬁve times, 5 min each at room temperature with agitation.

6. Follow the directions for the detection kit used to develop the blot. For the Immun-Star™ WesternC™ chemiluminescence kit (catalog #170-5070), mix 3 ml luminol/enhancer with 3 ml peroxide solution to make a 1x working solution for a 7 x 8.5 cm membrane. Incubate the membrane in the solution for 3–5 min. Prior to imaging, drain the excess substrate and place the membrane in a protective sleeve (such as plastic wrap) to prevent drying.

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Page 35

Table 12.1. Troubleshooting electrophoresis and detection with Mini-PROTEAN® gels. For more troubleshooting tips, refer to the relevant instrument instruction manuals or contact Technical Support.

Current is zero or less than expected, and samples do not migrate into gel

Troubleshooting

Problem Cause Solution

Tape at bottom of cassette not removed Remove tape

Insufﬁcient buffer in inner buffer chamber

Fill buffer chamber with running buffer

Insufﬁcient buffer in outer buffer chamber

Incorrect cassette orientation Ensure shorter plate is facing gasket

Electrical disconnection Check electrodes and connections

Gels run faster than expected Running buffer too concentrated or

incorrect

Gel temperature too high Do not exceed recommended running

Gels run more slowly than expected Companion running module left in tank

when running only 1–2 gels

Buffer leaking from inner chamber Incomplete gasket seal Wet gasket with running buffer before use

Improper assembly of the gel into the electrode/companion module

Bands “smile” across gel: band pattern curves upward at both sides of gel

Excessive heating of gel Check buffer composition

Insufﬁcient buffer Fill inner and outer chambers to ensure

Fill inner and outer chambers to ensure wells of the gels are completely covered

Check buffer composition

conditions

Remove companion running module

Top edge of short plate should ﬁt under notch at top of gasket

Top of short plate should touch green gasket

Do not exceed recommended running conditions

wells of gels are completely covered

Bands “smile” or “frown” within gel lanes

Protein load too high Load less protein

Sample or buffer preparation issues Minimize salts, detergents, and solvents

in sample preparation and sample loading buffers

Incorrect running conditions Set correct voltage

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Page 36

Mini-PROTEAN Precast Gels

Problem Cause Solution

Bands are skewed or distorted; lateral band spreading

Artifactual bands at 60 –70 kD

Poor resolution or fuzzy bands Sample volume is too high If possible, load a more concentrated

Too much salt in samples Remove salt from samples (dialysis,

precipitation, or other method)

Insufﬁcient or wrong sample buffer Check buffer composition and dilution

Sample precipitation Selectively remove predominant proteins

Dilute sample in sample buffer

Insoluble materials (for example, cell membranes) in samples

Skin keratin contamination Clean all dishware; wear gloves while

Diffuse sample loading zone Load sample with a syringe or gel loading

Centrifuge samples to remove particulates prior to sample loading

handling and loading gels

Filter all solutions (0.2–0.45 µm ﬁlter)

sample in a lower sample buffer volume

pipet tip

Sample diffusion during staining with Coomassie

Incompatible sample components Minimize salts, detergents, and solvents in

Expired gel Use gels before expiration date on cassette

Fix gel with 40% methanol, 10% acetic acid for 80 min prior to staining

sample preparation and loading buffers

Mini-PROTEAN® TGX Stain-Free™ Gels

Low sensitivity for proteins Low tryptophan content in proteins After activation and imaging, stain gel with

Bio-Safe™ Coomassie or similar to detect missing bands

Uneven sensitivity or fuzzy bands Gel was soaked in water or buffer prior

to activation and imaging

Bands are too light or missing from blot (membrane)

Standards are not visible Incorrect standards were used Use unstained standards; some prestained

Dye front at bottom of gels interferes with detection of proteins

Proteins transferred through membrane Use membrane with smaller pore size

Sample constituents present in gel interfering with imaging

If possible, activate and image gel immediately after electrophoresis

Decrease transfer time

Decrease voltage

standards are not detected by the imager. To monitor electrophoresis, use a 1:1 mixture of unstained and prestained standards

Dilute sample in gel running buffer prior to loading

Activate and image gel, rinse in ﬁxation solution for 30 min, and repeat imaging

Signal intensity on blot is lower than expected

Sample bands are faint relative to prestained standards

Trihalo compounds bound to tryptophan residues inhibit binding of some antibodies

Brightness of prestained standards can limit exposure times for sample bands

Blot gel without stain-free activation. If signal intensity is restored, use another (preferably polyclonal) antibody, if available

In Image Lab™ software, select Faint Bands to optimize exposure time or manually deﬁne longer exposure

Adjust transform to optimize contrast for fainter bands

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Page 37

This section contains abbreviated protocls (quick start guides) for the following electrophoretic techniques. Directions are for use of Mini-PROTEAN® precast gels and the Mini-PROTEAN® Tetra ce ll.

Quick Start Guides

SDS-PAGE using Mini-PROTEAN® TGX™ or Mini-PROTEAN® TGX Stain-Free™ precast gels

Native PAGE using Mini-PROTEAN TGX or Mini-PROTEAN TGX Stain-Free precast gels

Peptide analysis using Mini-PROTEAN Tris-Tricine gels

Nondenaturing PAGE of nucleic acids using Mini-PROTEAN TBE gels

Denaturing PAGE of nucleic acids using Mini-PROTEAN TBE-urea gels

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Page 38

Mini-PROTEAN Precast Gels

SDS-PAGE (Mini-PROTEAN TGX Gels)

Prepare Buffers

Running buffer (1x) Dilute 100 ml 10x stock (catalog #161-0732) with 900 ml diH2O.

Sample buffer Use Laemmli sample buffers: catalog #161-0737 or catalog #161-0747 (4x).

Prepare Gels and

Assemble Electrophoresis Cell

Remove the comb and tape from the gels, rinse wells, and assemble the electrophoresis cell.

Fill the inner and outer buffer chambers with running buffer.

Prepare and Load Samples

Component Reducing Nonreducing

Sample 5 μl 5 μl

Laemmli sample buffer, 2x

4.75 μl 5 μl

(catalog #161-0737)

β-Mercaptoethanol 0.25 μl —

Total volume 10 μl 10 μl

Heat samples at 90–100°C for 5 min (or at 70°C for 10 min).

Load the appropriate amount of sample on the gel.

Perform Electrophoresis

Connect the electrophoresis cell to the power supply and perform electrophoresis according to the conditions in the table.

Table A.1. Running conditions for SDS-PAGE in the Mini-PROTEAN Tetra cell. Standard conditions are constant 300 V.

100 V 200 V 300 V

Run time 85–95 min 30–40 min 15–20 min

Expected current (per gel)

Initial 15–20 mA 25–50 mA 55–75 mA

Final 5–10 mA 20–31 mA 45–70 mA

Expected temperature 25°C 25–35°C 30–45°C

Outer buf fer volume

1–2 Gels 2-gel mark 2-gel mark 4-gel mark 3–4 Gels 4-gel mark 4-gel mark 4-gel mark

Requires the PowerPac™ HV or PowerPac Universal power supply.

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Page 39

Instruction Manual and Application Guide

Native PAGE (Mini-PROTEAN TGX Gels)

Prepare Buffers

Running buffer (1x) Dilute 100 ml 10x stock (catalog #161-0734) with 900 ml diH2O.

Sample buffer Use native sample buffer (catalog #161-0738)

Prepare Gels and

Assemble Electrophoresis Cell

Remove the comb and tape from the gels, rinse wells, and assemble the electrophoresis cell.

Fill the inner and outer buffer chambers with running buffer.

Prepare and Load Samples

Component Volume

Sample 5 μl

Native sample buffer

5 μl

(catalog #161-0738)

Total volume 10 μl

Load the appropriate amount of sample on the gel.

Perform Electrophoresis

Connect the electrophoresis cell to the power supply and perform electrophoresis according to the conditions in the table.

Table A.2. Standard running conditions for native PAGE with one (1) Mini-PROTEAN TGX gel in the Mini-PROTEAN Tetra cell. Run conditions and times are approximate and assume a constant voltage of 200 V. When running more than one

gel, current will differ but temperature and run time should be close to those listed.

Current (mA) at 200 V Gel Initial Final Temperature Run Time

1 Gel (buffer to 2-gel mark)

7.5% 35–37 17–20 28–30°C 38 –40 min 10% 12%

4–15% 50–55 25–28 30–33°C 30–34 min 4–20% Any kD™

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Page 40

Mini-PROTEAN Precast Gels

Peptide Analysis (Mini-PROTEAN Tris-Tricine Gels)

Prepare Buffers

Running buffer (1x) Dilute 100 ml 10x stock (catalog #161-0744) with 900 ml diH2O.

Sample buffer (2x) 200 mM Tris-HCl, pH 6.8, 2% SDS, 40% glycerol, 0.04% Coomassie (catalog #161-0739) Brilliant Blue G-250, 2% ß-mercaptoethanol or 350 mM DTT (added fresh)

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.

Prepare and Load Samples

Component Reducing Nonreducing

Sample 5 μl 5 μl

Sample buffer

4.75 μl 5 μl

(catalog #161-0739)

β-Mercaptoethanol 0.25 μl —

Total volume 10 μl 10 μl

Heat samples at 90–100°C for 5 min or at 70°C for 10 min.

Load the appropriate amount of sample on the gel.

Perform Electrophoresis

Connect the electrophoresis cell to the power supply and perform electrophoresis according to the conditions in the table.

Table A.3. Running conditions for one (1) Mini-PROTEAN Tricine gel in the Mini-PROTEAN Tetra cell. Run conditions and times are approximate and assume a constant voltage of 100 V. When running more than one gel, current will differ.

16.5% Gels 10–20% Gels

Power conditions 100 V constant 100 V constant

Expected current (per gel)

Initial 65 mA 65 mA

Final 35 mA 35 mA

Run time 100 min 100 min

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Page 41

Instruction Manual and Application Guide

Nondenaturing Nucleic Acid PAGE (Mini-PROTEAN TBE Gels)

Prepare Buffers

Running buffer (1x) Dilute 100 ml 10x stock (catalog #161-0733) with 900 ml diH2O.

Sample buffer (5x) 50 mM Tris-HCl, pH 8.0, 5mM EDTA, 25% glycerol, 0.2% bromophenol (catalog #161-0767) blue, 0.2% xylene cyanole FF

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.

Prepare and Load Samples

Component Amount

Sample 8 μl

Sample buffer

2 μl

(catalog #161-0767)

Total volume 10 μl

Load the appropriate amount of sample on the gel.

Perform Electrophoresis

Connect the electrophoresis cell to the power supply and perform electrophoresis according to the conditions in the table.

Table A.4. Running conditions for nondenaturing nucleic acid PAGE with one (1) Mini-PROTEAN TBE gel in the Mini-PROTEAN Tetra cell. Run conditions and times are approximate and assume a constant voltage of 100 V. When running

more than one gel, current will differ.

5% Gels 10% Gels 15% Gels 4–20% Gels

Power conditions 100 V constant 100 V constant 100 V constant 100 V constant

Expected current (per gel)

Initial 15 mA 15 mA 15 mA 15 mA

Final 10 mA 10 mA 10 mA 10 mA

Run time 45 –60 min 60–75 min 75–90 min 90–105 min

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Page 42

Mini-PROTEAN Precast Gels

Denaturing Nucleic Acid PAGE (Mini-PROTEAN TBE-Urea Gels)

Prepare Buffers

Running buffer (1x) Dilute 100 ml 10x stock (catalog #161-0733) with 900 ml diH2O.

Sample buffer (5x) 89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.0, 12% Ficoll, (catalog #161-0768) 0.01% bromophenol blue, 0.02% xylene cyanole FF, 7 M urea

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.

Prepare and Load Samples

Component Amount

Sample 8 μl

Sample buffer

2 μl

(catalog #161-0768)

Total volume 10 μl

Load the appropriate amount of sample on the gel.

Perform Electrophoresis

Connect the electrophoresis cell to the power supply and perform electrophoresis according to the conditions in the table.

Table A.5. Running conditions for denaturing nucleic acid PAGE with one (1) Mini-PROTEAN TBE-urea gel in the Mini-PROTEAN Tetra cell. Run conditions and times are approximate and assume a constant voltage of 200 V. When running

more than one gel, current will differ.

10% Gels 15% Gels

Power conditions 200 V constant 200 V constant

Expected current (per gel)

Initial 15 mA 15 mA

Final 10 mA 10 mA

Run time 45–60 min 60–75 min

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Page 43

Buffers

Running Buffers

10x SDS-PAGE (1 L) 250 mM Tris, 1.92 M glycine, 1% SDS, pH 8.3

(catalog #161-0732) Tris base 30.3 g Glycine 144.1 g SDS 10 g diH2O to 1 L Do not adjust the pH (~pH 8.3)

10x Native PAGE (1 L) 250 mM Tris, 1.92 M glycine, pH 8.3

(catalog #161-0734) Tris base 30.3 g Glycine 144.1 g diH2O to 1 L Do not adjust the pH (~pH 8.3)

10x Tris-Tricine (1 L) 1 M Tris, 1 M Tricine, 1% SDS, pH 8.3 (catalog #161-0744) Tris base 121.1 g Tricine 179.2 g SDS 10 g diH2O to 1 L Do not adjust the pH (~pH 8.3)

10x TBE (1 L) 890 mM Tris, 890 mM boric acid, 20 mM EDTA

(catalog #161-0741) Tris base 107.8 g Boric acid 55.0 g EDTA 5.8 g diH2O to 1 L Do not adjust the pH (~pH 8.3)

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Page 44

Mini-PROTEAN Precast Gels

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 3.75 ml

50% Glycerol 15.0 ml

1.0% Bromophenol blue 0.3 ml

10% SDS 6.0 ml diH2O 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, 0.01% (catalog #161-0738) bromophenol blue

0.5 M Tris-HCl, pH 6.8 3.75 ml

50% Glycerol 24 ml

1.0% Bromophenol blue 0.3 ml

diH2O to 30 ml

2x Tricine (30 ml) 200 mM Tris-HCl, pH 6.8, 2% SDS, 40% glycerol, 0.04% (catalog #161-0739) Coomassie Brilliant Blue G-250, 2% β-mercaptoethanol (added fresh)

1.0 M Tris-HCl, pH 6.8 6.0 ml

100% Glycerol 12.0 ml 10% SDS 6.0 ml Coomassie Blue G-250 12.0 mg diH2O to 30 ml

 Add β-mercaptoethanol (20 µl to 980 µl sample buffer) before use.

5x Nucleic acid (10 ml) 50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 25% glycerol, 0.2%

(catalog #161-0767) bromophenol blue, 0.2% xylene cyanole FF

Tris base 78.8 mg 50% Glycerol 5 ml EDTA 14.6 mg

1.0% Bromophenol blue 2.0 ml

Xylene cyanole FF 20.0 mg diH2O to 10 ml

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Page 45

Instruction Manual and Application Guide

TBE-urea (30 ml) 89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.0, 12%

(catalog #161-0768) Ficoll, 0.01% bromophenol blue, 0.02% xylene cyanole, Store at 4°C 7 M urea

Tris base 0.32 g Boric acid 0.165 g EDTA 17.5 mg Ficoll 3.6 g Bromophenol blue 3 mg Xylene cyanole FF 6 mg Urea 12.6 g diH2O to 30 ml

Buffer Components

0.5 M Tris-HCl, pH 6.8 (1 L) Tris base 60.6 g (catalog #161-0799) diH2O ~900 ml Store at 4°C Adjust to pH 6.8 with HCl diH2O to 1 L

10% SDS (250 ml) SDS 25.0 g (catalog #161-0416) di H2O to 250 ml

1.0% Bromophenol blue (10 ml) Bromophenol blue 100 mg (10 g powder, catalog #161-0404) diH2O to 10 ml

Blotting Buffers

Towbin buffer (1 L) 25 mM Tris, 192 mM glycine, 20% methanol

Dissolve: Tris base 3.03 g Glycine 14.4 g diH2O 500 ml

Then add: Methanol 200 ml diH2O to 1 L

Alternatively, use: 10x Tris/glycine (catalog #161-0734) 100 ml Add 200 ml methanol and diH2O to 1 L as above

Tris-buffered saline with 20 mM Tris, 500 mM NaCl, 0.05% Tween 20 Tween (TTBS, 1 L) Tris base 2.4 g

NaCl 29.2 g 10% Tween 20 5.0 ml diH2O to 1 L

Alternatively, use: 10x TBS (catalog #170-6435) 100 ml 10% Tween 20 (catalog #166-2404) 5 ml diH2O 895 ml

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Page 46

Bulletin # Title

10007296 Mini-PROTEAN® Tetra Cell Instruction Manual

4006191 Mini-PROTEAN® 3 Dodeca™ Cell Instruction Manual

1703930 Mini Trans-Blot® Instruction Manual

10020688 Trans-Blot® Turb o™ Blotting System Instruction Manual

4006066 Trans-Blot® SD Semi-Dry Transfer Cell Quick Reference Guide

1703940 Trans-Blot SD Semi-Dry Transfer Cell Instruction Manual

10014472 Ge l D oc™ EZ System Installation Guide

10019634 Stain-Free™ Sample Tray Instruction Manual

10022469 ChemiDoc™ MP System with Image Lab Software Instruction Manual

5871 Mini-PROTEAN® TGX™ Precast Gels Product Information Sheet

5535 Mini-PROTEAN Tetra Cell Brochure

Related Literature

2895 Protein Blotting Guide

3133 Molecular Weight Determination by SDS-PAGE

3144 Using Precision Plus Protein™ Standards to Determine Molecular Weight

6088 Bio-Rad V3 Western Workﬂow™ Brochure

1939 Blotting Membrane Brochure

6116 Immun-Blot® LF PVDF Membranes Product Information Sheet

2032 Western Blotting Detection Reagent Brochure

2317 Ready-to-Run Buffers and Solutions Brochure

2414 The Little Book of Standards

40 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com

Page 47

Mini-PROTEAN® TGX™ Precast Gels

10 Gels per Box

7. 5% 456 -10 29 45 6-1023 4 56-1024 456 -1025 4 56-1026 4 56-1021

10% 456-1039 456-1033 456 -103 4 45 6 -10 35 456 -103 6 4 56-1031

12% 456-1049 456 -10 4 3 4 56-1044 456-1045 456-1046 4 56-1041

4–15% 4 56-1089 4 5 6-10 83 456 -108 4 45 6 -10 85 456 -108 6 4 56-1081

4–20% 456 -109 9 4 56-1093 456-1094 4 56-1095 4 56-1096 4 56-1091

8-16% 4 5 6 -1109 4 56-110 3 4 5 6 -1104 4 5 6 -1105 4 5 6 -1106 4 5 6 -1101

Any kD

Mini-PROTEAN® TGX Stain-Free™ Precast Gels

10 Gels per Box

7. 5% 456-8029 456-8023 456-8024 456-8025 456-8026 456-8021

10% 456-8039 456-8033 456-8034 456-8035 456-8036 456-8031

12% 4–15% 4-20% 8-16%

Any kD 45 6-8129 456 -8123 45 6-8124 456-8125 456 -8126 546-8121

™

Ordering Information

8+1 Wel l 10-Well 12-Well 15-Well IPG/Prep

7 cm IPG strip

(30 µl/well) (30 µl/well) (50 µl/well) (20 µl/well) (15 µl/well) (450 µl/well)

456-9039 456-9033 456-9034 456-9035 456-9036 456-9031

8+1 Wel l 10-Well 12-Well 15-Well IPG/Prep

7 cm IPG strip

(30 µl/well) (30 µl/well) (50 µl/well) (20 µl/well) (15 µl/well) (450 µl/well)

456-8049 456-8089 456-8099

456- 8109

456-8043 456-8083 456-8093 456- 8103

456-8044 456-8084 456-8094 456- 8104

456-8045 456-8085 456-8095 456- 8105

456-8046 456-8086 456-8096 456- 8106

456 -80 41 456-8081 456-8091 456- 8101

Note: Mini-PROTEAN TGX and TGX Stain-Free gels are available in 10-packs (catalog numbers listed) or 2-packs (add an "S" to the end of the catalog number listed).

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Page 48

Mini-PROTEAN Precast Gels

Mini-PROTEAN Tris-Tricine Precast Gels

2 Gels per Box

10-Well 15-Well

(30 µl/well) (50 µl/well) (15 µl/well)

16.5% 456-3063 456-3064 456-3066

10–20% 4 5 6 -3113 4 5 6 - 3114 4 5 6 - 3116

Mini-PROTEAN TBE Precast Gels

2 Gels per Box 2 Gels per Box

10-Well 15-Well 10-Well 15 -Well

(30 µl/well) (50 µl/well) (15 µl/well) (30 µl/well) (50 µl/well (15 µl/well)

5% 456-5013 456-5014 456-5016 15% 456-5053 456-5054 456-5056

10% 456-5033 456-5034 456-5036 4–20% 456-5093 456-5094 456-5096

Catalog # Description

Protein Standards

161-0363 Precision Plus Protein™ Unstained Standards (10–250 kD), 1 ml, 100 applications

Mini-PROTEAN TBE-Urea Precast Gels

2 Gels per Box

10-Well 15-Well

(30 µl/well) (15 µl/well)

10% 456-6033 456-6036

15% 456-6053 456-6056

161-0373 Precision Plus Protein All Blue 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™ Standards (10–250 kD), 250 μl, 50 applications

161-0377 Precision Plus Protein Dual Xtra Standards (2–250 kD), 500 μl, 50 applications

161-0385 Precision Plus Protein WesternC Pack (10–250 kD), 50 applications

161-0317 SDS-PAGE Standards, broad range, 200 μl

Equipment

165-8004 Mini-PROTEAN® Tetra Cell for Ready Gel™ Precast Plates (4-gel system)

165-8005 Mini-PROTEAN Tetra Cell for Ready Gel Precast plates (2-gel system)

165-4100 Mini-PROTEAN® 3 Dodeca™ Cell

170-3930 Mini Trans-Blot® Electrophoretic Transfer Cell

170-3940 Trans-Blot® SD Semi-Dry Electrophoretic Transfer Cell

170 -4155 Tran s-B lot® Turb o™ Transfer Starter System

164-5050 PowerPac™ Basic Power Supply

164-5052 PowerPac HC High Current Power Supply

170-8270 Gel Doc™ EZ Imaging System

170-8274 Stain-Free™ Sample Tray

170-8280 ChemiDoc™ MP Imaging System with Image Lab™ Software

42 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com

Page 49

Instruction Manual and Application Guide

Catalog # Description

Premixed Running Buffers

161-0732 10x Tris/Glycine/SDS, 1 L

161-0772 10x Tris/Glycine/SDS, 5 L

161-0734 10x Tris/Glycine, 1 L

161-0744 10x Tris/Tricine/SDS, 1 L

161-0733 10x Tris/Boric Acid/EDTA, 1 L

161-0770 10x Tris/Boric Acid/EDTA, 5 L

Premixed Sample Buffers

161-0737 Laemmli Sample Buffer, 30 ml*

161-0747 4x Laemmli Sample Buffer, 10 ml*

161-0738 Native Sample Buffer, 30 ml

161-0739 Tricine 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, 100 g

161-0416 SDS Solution, 10% (w/v), 250 ml

166-2404 10% Tween 20, 5 ml

170-6404 Blotting-Grade Blocker, 300 g

161-0710 2-mercaptoethanol, 25 ml

161-0611 Dithiothreitol, 5 g

161-0404 Bromophenol Blue, 10 g

Blotting Membranes

162-0212 0.2 μm Nitrocellulose/Filter Paper Sandwich, 7 x 8.5 cm, 20 pack

162-0213 0.2 μm Nitrocellulose/Filter Paper Sandwich, 7 x 8.5 cm, 50 pack

162-0214 0.45 μm Nitrocellulose/Filter Paper Sandwich, 7 x 8.5 cm, 20 pack

162-0215 0.45 μm Nitrocellulose/Filter Paper Sandwich, 7 x 8.5 cm, 50 pack

162-0216 Sequi-Blot™ PVDF/Filter Paper Sandwich, 7 x 8.5 cm, 20 pack

162-0217 Sequi-Blot PVDF/Filter Paper Sandwich, 7 x 8.5 cm, 50 pack

170-4156 Trans-Blot Turbo Midi PVDF Transfer Packs

170-4158 Trans-Blot Turbo Midi Nitrocellulose Transfer Packs

162-0260 Immun-Blot® Low Fluorescence PVDF/Filter Paper Sets, 10 pack

162-0261 Immun-Blot Low Fluorescence PVDF/Filter Paper Sets, 20 pack

162-0264 Immun-Blot Low Fluorescence PVDF Membrane

May require addition of 2-mercaptoethanol or DTT

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Page 50

Mini-PROTEAN Precast Gels

Catalog # Description

Total Protein Gel and Blot Stains

161-0786 Bio-Safe™ Coomassie Stain, 1 L

161-0400 Coomassie Brilliant Blue R-250, 10 g

161-0436 Coomassie Brilliant Blue R-250 Stain Solution, 1 L

161-0438 Coomassie Brilliant Blue R-250 Destain Solution, 1 L

161-0443 Silver Stain Kit

161-0449 Silver Stain Plus™ Kit

170-6527 Colloidal Gold Total Protein Stain, 500 ml

161-0440 Zinc Stain and Destain Kit

170-3127 SYPRO Ruby Protein Blot Stain, 200 ml

161-0491 Flamingo™ Fluorescent Gel Stain (10x), 100 ml

161-0496 Oriole™ Fluorescent Protein Gel Stain (1x), 1 L

Immunoblot Detection Reagents

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™ AP Substrate Pack

For additional product sizes, please refer to the Bio-Rad catalog or website.

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Page 51

Instruction Manual and Application Guide

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Page 52

Life Science Group

Sig 12111658100 Rev F US/EG

Bio-Rad Laboratories, Inc.

Web site ww w.bio-rad.com USA 800 424 6723 Australia 61 2 9914 2800 Austria 01 877 89 01 Belgium 09 385 55 11 Brazi l 55 11 5044 5699 Canada 905 364 3435 China 86 21 6169 8500 Cz ech Republic 420 241 430 532 Denmar k 44 52 10 00 Finland 09 804 22 00 France 01 47 95 69 65 Germany 089 31 884 0 Greece 30 210 9532 220 Hon g Kong 852 2789 3300 Hungar y 36 1 459 6100 India 91 124 4029300 Israel 03 963 6050 Italy 39 02 2 16091 Japan 03 6361 7000 Korea 82 2 3473 4460 Mex ico 52 555 488 7670 The Netherlands 0318 540666 New Zealand 64 9 415 2280 Nor way 23 38 41 30 Poland 48 22 331 99 99 Portugal 351 21 472 7700 Russia 7 495 721 14 04 Singapore 65 6415 3188 South Africa 27 861 246 723 Spain 34 91 590 5200 Sweden 08 555 1270 0 Switzerland 061 7 17 95 55 Taiwan 886 2 2578 7189 Thailand 800 88 22 88 United Kingdom 020 8328 200 0

Bio-Rad Mini-PROTEAN TGX Precast Gels for 2-D Electrophoresis User Manual

Specifications and Main Features

Frequently Asked Questions

User Manual