tgx gels instructions

8/14/2019 TGX Gels Instructions

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Mini-PROTEAN

Precast GelsInstruction Manual

and

Application Guide

For technical support, call your local Bio-Rad office, or in the U.S., call 1-800-4BIORAD (1-800-424-6723)


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Table of Contents

Section 1 General Information ...................................................................11.1 Introduction.............................................................................................1

1.2 Mini-PROTEAN Precast Gel Specifications.............................................21.3 Important Notes ......................................................................................3

1.4 Mini-PROTEAN Comb Configurations.....................................................3

Section 2 Setup and Basic Operation........................................................32.1 Required Materials..................................................................................32.2 Mini-PROTEAN Precast Gel Set-Up Overview........................................4

2.3 Assembling the Mini-PROTEAN Tetra Cell Electrophoresis Module ........5

Section 3 SDS-PAGE...................................................................................63.1 Introduction.............................................................................................63.2 Mini-PROTEAN TGX Gel Composition....................................................73.3 Mini-PROTEAN TGX Gel Selection Guide ..............................................7

3.4 SDS-PAGE Buffers .................................................................................8

3.5 Sample Preparation ................................................................................83.6 Running Conditions.................................................................................8

Section 4 Native PAGE................................................................................84.1 Introduction.............................................................................................84.2 Native PAGE Buffers...............................................................................8

4.3 Sample Preparation ................................................................................84.4 Running Conditions.................................................................................8

Section 5 Buffers.........................................................................................9

Section 6 Total Protein Gel Stains for SDS-PAGE and Native PAGEDetection...................................................................................10

Section 7 Troubleshooting........................................................................11

Appendix A Stock Solutions.........................................................................13

Appendix B Total Protein Blot Stains...........................................................14

Appendix C Related Literature .....................................................................14

Appendix D Ordering Information ................................................................15D.1 Mini-PROTEAN TGX Precast Gels .................................................15D.2 Premixed Running and Sample Buffers ..........................................15

D.3 Individual Reagents ........................................................................15D.4 Total Protein Gel and Blot Stains....................................................16

D.5 Immunoblot Detection.....................................................................17D.6 Immunoblot Detection Reagents.....................................................17D.7 Blotting Membranes........................................................................18

D.8 Protein Standards...........................................................................18D.9 Equipment ......................................................................................18


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Section 1General Information

1.1 Introduction

Mini-PROTEANprecast gels greatly simplify polyacrylamide gel electrophoresis. They arespecifically designed for use with the Mini-PROTEAN Systems (Mini-PROTEAN Tetra,Mini-PROTEAN 3, and Mini-PROTEAN Dodeca Cells).

Mini-PROTEAN precast gels come ready to use with pre-formed sample wells and a stackinglayer. Each Mini-PROTEAN cassette is 8.5 cm x 10 cm (H x W) and 4.0 mm thick. Gel dimension

is 7.2 cm x 8.6 cm (H x W) and 1.0 mm thick. Each gel is individually packaged in a leak proofstorage pouch with gel buffer containing 0.02% sodium azide.

The migration pattern of proteins on Mini-PROTEAN TGX precast gels is similar to thatobserved with standard Laemmli Tris-HCl gels. Mini-PROTEAN TGX precast gels are run using

standard Laemmli sample buffer and Tris-Glycine-SDS running buffer. The precast gels containno sodium dodecyl sulfate (SDS) and can therefore be used for either sodium dodecyl sulfate

polyacrylamide gel electrophoresis (SDS-PAGE) or native gel electrophoresis depending uponthe sample buffer and the running buffer used.

Advantages of Mini-PROTEAN TGX precast gels:

Increased stability and long shelf life up to 12 months

Laemmli-like separation pattern

Exceptionally straight lanes and sharp bands

No need for special, expensive buffers

Superior staining quality

No gel foot to remove prior to blotting

Bottom open cassette that unlocks with four easy clicks

The Mini-PROTEAN Tetra cell runs both hand cast gels and Mini-PROTEAN TGX precastgels interchangeably. The cell can run from one to four gels, and the mini tank is compatible

with other Bio-Rad electrode modules for tank blotting.

The Mini-PROTEAN 3 cell runs both hand-cast and Mini-PROTEAN TGX precast gels.

The cell can run one or two gels, and the mini tank is compatible with other Bio-Rad electrodemodules for tank blotting, 2-D electrophoresis and electroelution.

The Mini-PROTEAN 3 Dodeca cell is a multi-cell for high through-put system gelelectrophoresis. It can run up to 12 identical polyacrylamide gels simultaneously. The Dodeca

cell includes clamping frames, buffer dams, and a drain line.

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1.2 Mini-PROTEAN Precast Gel Specifications

Gel material Polyacrylamide

Gel dimensions 7.2 x 8.6 cm (H x W)

Gel thickness 1.0 mm

Resolving gel height 6.2 cm

Cassette dimensions 8.5 x 10 cm (H x W)

Cassette material Styrene copolymer

Comb material Polycarbonate

Total running buffer volume 700 ml for 2 gels, 1,000 ml for 4 gels(Mini-PROTEAN Tetra Cell & Mini-PROTEAN 3)

Storage conditions Store flat between 2C and 8C; DO NOT FREEZE

Mini-PROTEAN Tetra Cell Specifications

Casting stand Polycarbonate

Pin, retaining ring and spring Stainless steel

Casting frames PolysulfoneGray gaskets Thermoplastic rubber (gray)

Electrode assembly Glass filled polybutylene terephthalate

Electrodes Platinum wire, 0.010 diameter

Gasket, electrode inner core Silicone rubber (green)

Tank and lid Polycarbonate

Sample loading guides Delrin

Combs Polycarbonate

Mini-PROTEAN 3 Cell Specifications

Electrode assembly Glass-filled liquid crystal polymer

Electrodes Platinum wire, 0.010 diameterGasket, electrode inner core Silicone rubber (green)

Tank and lid Molded polycarbonate

Sample loading guides Delrin

Combs Polycarbonate

Mini-PROTEAN 3 Dodeca Cell Specifications

Tank and lid Acrylic

Clamping frame Polycarbonate and liquid crystal polymer

Upper electrode holder Polycarbonate with 109 mm (4.3) platinum wire

Lower electrode assembly Polycarbonate with 89 mm (3.5) platinum wire

Drain line Tygon tubing

Drain line connectors Delrin

Cooling coil Acrylic

Cooling coil connector tubing Tygon

Maximum buffer volume 4.4 L

Minimum buffer volume 3.4 L

Overall size 41.5 x 15 x 16.2 cm (L x W x H)

Safety limits 300 V, 150 W

Weight 5 kg (11 lb)

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1.3 Important Notes (See Appendix C for Related Literature)

Mini-PROTEAN Tetra and Dodeca cell components are not compatible with acetone or

ethanol. Use of organic solvents voids all warranties.

Each Mini-PROTEAN precast gel should be used shortly after it is removed from the

storage pouch.

It is not advisable to run more than one gel type in the same apparatus at the same

time. The different gel percentages will have different conductivity and therefore differentrun rates.

When running 1 or 2 gels in the Tetra cell, use the electrode assembly (with the bananaplugs), not the companion running module (without the banana plugs). When running

3 or 4 gels, both the electrode assembly and the companion running module must beused.

When running 1 or 2 gels only, DO NOT place the companion running module in thetank. Doing so will cause excessive heat generation and degrade the quality of the

electrophoretic separation.

Improper storage of Mini-PROTEAN precast gels can produce numerous artifacts. Gels

should be stored flat between 2C and 8C. Avoid freezing or prolonged storage above8C. If you suspect your gels have been stored improperly, THEY SHOULD BE

DISCARDED.

Do not attempt to lock the green arms of the electrode assembly without first ensuring

that the gel cassettes are correctly aligned against the notches on the green gaskets ofthe module. To prevent the gels from shifting during the locking step, firmly and evenlygrip them in place against the core of the module (see Figure 2c and 2e).

1.4 Mini-PROTEAN Comb Configurations

Mini-PROTEAN TGX Gel

Comb type Well volume

10 well 30 l

15 well 15 l

IPG 7 cm ReadyStrip IPG strip

Section 2Setup and Basic Operation

2.1 Required Materials

Clean Mini-PROTEANTetra cell tank

Electrophoresis module: to run 1 or 2 gels, use the electrode module. To run 3 or 4gels, use the electrode module and companion module

PowerPac power supply or equivalent

Sample buffer

Running buffer (700 ml for 2 gels; 1,000 ml for 4 gels)

Mini-PROTEAN precast gels

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Fig. 1. Mini-PROTEAN Precast Gel Cassette.

2.2 Mini-PROTEAN Precast Gel Set Up Overview

1. Remove Comb: Position both thumbs on the ridges of the comb. Remove the comb by

pushing upward in one smooth continuous motion.

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

3. Rinse Wells: Use a syringe wash bottle or a disposable transfer pipette to rinse the wellswith running buffer. Straighten the sides of the wells, if necessary.

4. Run Gel: Assemble the cassette into the running module of the Mini-PROTEAN system.Add running buffer to the inner and outer chambers. Prepare the samples in sample buffer

and load the samples into the wells. Run the gel at 200 V until the dye front reaches theline on the bottom of the gel cassette (approximately 3040 min). At the completion of therun, disconnect the cell and remove the cassette.

5. Open Cassette: Align the arrow on the opening key with the arrows marked on thecassette. Insert the key between the cassette plates at all 4 locations and apply downward

pressure to break each seal. Do not twist the lever. Gently pull apart the two platesbeginning from the top of the cassette.

6. Remove Gel: Gently remove the gel from the cassette.

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Fig. 2. Assembling the Mini-PROTEAN Tetra Cell Electrphoresis Module.

2.3 Assembling the Mini-PROTEAN Tetra Cell Electrophoresis Module

1. Set the electrode assembly to the open position on a clean flat surface (see Figure 2a)

2. Place the first gel cassette (with the short plate facing inward) onto the gel supports; gelsupports are molded into the bottom of the electrode assembly. There are two supportson each side of the electrode assembly. Note that the gel will now rest at a 30 angle,

tilting away from the center of the electrode assembly. Use caution when placing thefirst gel, making sure that the electrode assembly remains balanced and does not tip

over. Place the second gel or buffer dam on the other side of the electrode assembly,again by resting the gel on the supports. At this point there will be two gels resting at a

30 angle, one on either side of the electrode assembly, tilting away from the center ofthe frame (see Figure 2b). It is critical that gel cassettes be placed into the electrodeassembly with the short plate facing inward to form the inner buffer chamber. The elec-

trode assembly requires two gels to create a functioning assembly; if an odd number ofgels (1 or 3) is being run, you must use the buffer dam to complete the assembly (see

Figure 2b).

3. Using one hand, 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 thegreen gasket (Figure 2e).

4. While gently squeezing the gel cassettes or a gel cassette and a buffer dam against the

green gaskets with one hand (keeping constant pressure and both gels firmly held inplace), slide the green arms of the clamping frame over the gels, locking them into

place (see Figure 2c).

5

2a 2b 2c

2d 2f

2e

Notch

GelCassette

Short Plate

Long Plate

Gel Support

Gasket

ClampingFrame


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5. The wing clamps of the electrode assembly lift each gel cassette up against the notchin the green gasket, forming a seal (Figure 2d). Check again to make certain that theshort plates sit just below the notch at the top of the green gasket (Figure 2e). Place the

assembled electrophoresis module into the tank (Figure 2f) and fill the buffer chambers.At this point, the sample wells can be washed out with running buffer, if this was not

done earlier, and the sample can be loaded. If running more than 2 gels, repeat steps2a2d with the companion running module.

Section 3SDS-PAGE

3.1 Introduction

Mini-PROTEANTGX precast gels provide a versatile system for sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), a gel electrophoretic technique

that separates proteins according to their molecular weight.

SDS-PAGE relies on a discontinuous buffer system. Two ions of differing electrophoretic

mobility (glycinate and chloride) form a moving boundary when voltage is applied. Proteinshave an intermediate mobility, causing them to concentrate, or stack, into a narrow zone atthe beginning of electrophoresis. As the boundary moves through the gel, the sieving effect

of the polyacrylamide gel matrix causes different proteins to move at different rates. Thestacking 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 intosharp bands prior to separation.

Protein samples for SDS-PAGE are prepared using SDS and a thiol reductant, usually2-mercaptoethanol or dithiothreitol (DTT). SDS forms complexes with proteins giving them

a rod like shape and similar charge to mass ratio. The reductant cleaves disulfide bondsbetween and within proteins allowing complete denaturation and dissociation. Heat

treatment in the presence of SDS and reductant effectively eliminates the effects of 2 and 3

protein structure and native charge on electrophoretic mobility, so the migration distancedepends primarily on molecular weight. Molecular mass is determined by plotting the

logarithm of protein molecular mass vs. the relative mobility (Rf) of the protein (R

f= dis-

tance migrated by protein/distance relative to the dye front of the protein). Refer to tech

notes 3133 and 3144.

Mini-PROTEAN TGX precast gels are prepared without SDS. Although gels for

SDS-PAGE have historically been cast with SDS in the gel, high quality SDS-PAGEseparations are obtained in gels lacking SDS, provided that the sample buffer and running

buffer contain sufficient SDS to maintain SDS saturation during electrophoresis. Therecommended concentrations of SDS are >1% in the sample buffer and 0.1% in the running

buffer. The absence of SDS in the gel itself provides additional flexibility, as the gels mayalso be used for native electrophoresis (see Section 4).

Mini-PROTEAN TGX precast gels differ from the standard Laemmli system (Tris-HClSDS-PAGE) gels due to a proprietary modification to their formulations that provides thegels with extended shelf life and improved separation characteristics. They are designed to

be run using standard Laemmli sample and running buffers. No additional special buffers orreagents are required.

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3.2 Mini-PROTEAN TGX Precast Gel Composition

Mini-PROTEAN TGX gels are comprised of polyacrylamide with a bisacrylamide cross

linker. Each gel has a 4% polyacrylamide stacking layer extending approximately 5 mmfrom the bottom of the loading well to the top of the resolving gel. The proprietary gel

formulation provides a shelf life of 12 months and improved separation characteristics.The gel is packaged with storage buffer of the same composition with additional 0.02%

sodium azide as a preservative.

3.3 Mini-PROTEAN TGX Precast Gel Selection Guide

Mini-PROTEAN TGX gels are available in a wide selection of single percentages and

gradients for the separation of proteins by SDS-PAGE.

Gel Selection Guide

Gel % Optimal Sample Running Run Conditions* RunType Separation Range Buffer Buffer Voltage/Current** Time***

TGX 7.5 40200 kD SDS-PAGE SDS-PAGE 200 V constant 38 minsample buffer running Starting current

buffer (per gel): 37 mAFinal current(per gel): 23 mA

TGX 10 30150 kD SDS-PAGE SDS-PAGE 200 V constant 38 minsample running Starting currentbuffer buffer (per gel): 37 mA

Final current(per gel): 23 mA


Final current

(per gel): 23 mA





TGX Any kD****10200 kD SDS-PAGE SDS-PAGE 200 V constant 28 minsample running Starting current

buffer buffer (per gel): 50 mAFinal current(per gel): 33 mA

*This may vary depending on water and buffer conductivity, which may vary from one lab setting to the next.

**Current should be multiplied by the number of gels being run.

***Approximate time required for dye front to reach the line at the bottom of the cassette.

****Any kD is a unique single percentage formulation that provides a broad separation range and short running time.

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3.4 SDS-PAGE Buffers

See Section 5 for buffer recipes.

3.5 Sample Preparation

The appropriate concentration of sample depends on the load volume and the detectionmethod used. (See Section 6 for approximate stain sensitivities). Add 50 l 2-mercaptoethanolper 950 l of sample buffer for a final concentration of 5% 2-mercaptoethanol, or 710 mM.

As an alternative, DTT may be used at a final concentration of 350 mM (54 mg/ml). Dilute1 part sample with at least 1 part sample buffer with added reductant. Heat the mixture at

95C for 5 min.

3.6 Running Conditions

Run gels at 200 V constant voltage until the dye front reaches the line near the bottom

edge of the gel cassette. Approximate run times will vary between 28 and 38 min dependingon the gel type (see Section 3.3).

Section 4Native PAGE

4.1 Introduction

Mini-PROTEANTGX gels are made without SDS, allowing native separations using

SDS- and reductant-free sample and running buffers. The nonreducing and nondenaturingenvironment of native PAGE allows protein separation with retention of biological activity.Native PAGE can also be used to resolve multiple protein bands when molecular mass

separation by SDS-PAGE would reveal only one.

Native PAGE uses the same moving boundary described in Section 3.1. Proteins are

prepared in nonreducing nondenaturing sample buffer, which maintains the proteins

secondary structure and native charge density. Protein mobility depends on the size andshape of the protein as well as its molecular weight and net charge. Native PAGE is thereforenot suitable for molecular weight determination.

4.2 Native PAGE Buffers

See Section 5 for buffer recipes.

4.3 Sample Preparation

Determine the desired protein concentration and load volume of your sample based on

the detection method used. (See Section 6 for approximate stain sensitivities). Proteins canbe separated using a standard protocol, following dilution of the sample with an equal

volume of Native Sample Buffer (see Section 5, DO NOT HEAT SAMPLES). Strongly basicproteins (pl >8.5) will have a net positive charge and will not enter a native PAGE TGX gel.

4.4 Running Conditions

See Section 3.3.

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Section 5Buffers (see Appendix A for Stock Solutions)

Name Working Concentration Notes Pre-Mixed Alternative

SDS-Pagerunning buffer 1X25 mM Tris base192 mM glycine0.1% (w/v) SDS

Running buffer shouldbe ~ pH 8.3. Do notadjust the pH

10x Tris/Glycine/SDS, 1 L,161-073210x Tris/Glycine/SDS, 5 L cube,161-0772

SDS-PAGE

sample buffer2X62.5 mM Tris HCl, pH 6.82% (w/v) SDS25% (v/v) glycerol0.01% (w/v) Bromophenol Blue5% (v/v) 2-mercaptoethanol or350 mM DTT (added fresh)

Dilute 1 part samplewith 1 part samplebuffer. More samplebuffer can be added ifnecessary. 1 partsample to 2 partssample buffer dilutionalso works. Drysamples can bedissolved directly intothe sample buffer

Laemmli sample buffer, 30 ml,161-0737

Native PAGErunning buffer

workingconcentration

25 mM Tris Base192 mM glycine

Running buffer shouldbe ~ pH 8.3. Do not

adjust the pH

10x Tris/Glycine, 1 L,

10x Tris /Glycine, 5

L cube,161-0771

Native PAGEsample buffer

62.5 mM Tris-HCl, pH 6.840% glycerol0.01% Bromophenol Blue

Native sample buffer, 30 ml,161-0738

-

-

-

161-0734

Dilute 1 part samplewith 1 part samplebuffer. More samplebuffer can be added ifnecessary. 1 partsample to 2 partssample buffer dilutionalso works. Drysamples can bedissolved directly intothe sample buffer

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Section 6Total Protein Gel Stains for SDS-PAGE and NativePAGE Detection

Method Sensitivity OptimalProtein

Load

Advantages Disadvantages Imaging InstructionManual

Number

CoomassieBlue R-250

3647 ng ~0.5g/band

Laboratorystandard

Requires MeOH Photographywith white light ortransmissiondensitometry(Gel Doc orGS-800)

Consultscientificliterature

Bio-SafeCoomassiestain

828 ng ~0.5g/band

Nonhazardous Photographywith white light ortransmissiondensitometry(Gel Doc orGS-800)

4307051

Zinc stain 612 ng ~0.2g/band

High-contrast,fast, reversiblesta

in

Negative stain,must be

photographed,SDS-PAGE

only

Photographywith white light or

tra

nsmi

ssiondensitometry

(Gel Doc or GS-800)

4006082

SilverStain Pluskit

0.61.2 ng ~0.01g/band

Simple,robust, massspectrometrycompatible

Does not stainGlycoproteinswell

Photographywith white light ortransmissiondensitometry(Gel Doc orGS-800)

LIT442

Silver stain 0.61.2 ng ~0.01g/band

Stainscomplexproteins, i.e.,glycoproteins,andlipoproteins

Not massspectrometrycompatible

Photographywith white light ortransmissiondensitometry(Gel Doc orGS-800)

LIT34

DodecaSilver StainKit

0.51.2 ng ~0.1g/band

Convenientstaining for alarge numberof gels

Photographywith white light ortransmissiondensitometry(Gel Doc or GS-800)

4110150

SYPRORuby proteingel stain

110 ng ~0.2g/band

Broaddynamicrange, simplerobust protocol

Requiresimaginginstrument formaximumsensitivity

Fluorescencevisualization withUV trans-illumination orlaser scanning

4006173

FlamingoFluorescent

0.250.5 ng ~0.01g/band

Broaddynamicrange, massspeccompatible

Requiresimaginginstrument formaximumsensitivity

Fluorescencevisualizationwith UV trans-illumination orlaser scanning(best option)

10003321

OrioleFluorescentprotein gelstain

0.5 ng ~0.2g/band

HighsensitivityBroaddynamic range

Will not workwith visibleexcitation

Fluorescencevisualization withUV transil-lumination (GelDoc, Chemi Doc)

10017295

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Section 7Troubleshooting

Problem Cause Solution

Current is zero or less than Tape at the bottom of Remove tapeexpected and samples do not the cassette notmigrate into gel removed

Insufficient buffer in inner Fill buffer chamber withbuffer chamber 700 ml running buffer

Insufficient buffer Make sure the innerin outer buffer and outer buffer

chamber chambers aresufficiently filled to

ensure that the wells ofthe gel are completelycovered

Electrical disconnection Check electrodes and

connections

Bands smile across gel, Excess heating of gel Check buffer composition

band pattern curves upward Do not exceedat both sides of the gel recommended running

conditions

Insufficient buffer Make sure the inner

and outer bufferchambers aresufficiently filled to

ensure that the wells of

the gel are completelycovered

Smiling or frowning bands Overloaded proteins Load less protein

within the gel lane Sample Consider minimizingpreparation/buffer salts, detergents and

issues solvents in samplepreparation and sample

buffer

Running speed Check to make sure the

correct voltage hasbeen set

Skewed or distorted bands, Excess salt in samples Remove salts fromlateral band spreading sample by dialysis or

desalting column priorto sample preparation

Insufficient sample Check bufferbuffer or wrong composition andformulation dilution instructions

Vertical streaking Overloaded samples Dilute sample

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Problem Cause Solution

Vertical streaking Sample precipitation Selectively removepredominant protein inthe sample

Dilute sample in more

sample buffer

Insoluble materials Centrifuge samples toin the samples remove particulates(e.g., membranes) prior to sample loading

Gels run faster than Running buffer is too Check bufferexpected concentrated and gel composition

temperature is too high Incorrect running buffer

type is used

Artifact bands at ~6070 kD Possible skin keratin Thoroughly clean allcontamination dishware and wear

gloves while handlingand loading gel

Filter all solutionsthrough 0.2 m or0.45 m filter

Leaking from inner Incomplete gasket seal Wet the gasket withbuffer chamber running buffer before

use

Improper assembly of Check that the top edgethe gel into the of the short plate fitselectrode/companion under the notch at theassembly top of the gasket

Make sure that the topof the short plate istouching the green

gasketPoor resolution High sample volume If possible, load a moreor fuzzy bands concentrated sample

in a lower volume ofsample buffer

Diffuse sample loading Load sample withzone syringe or gel loading

pipette tips

Sample diffusion during Fix gel with 40%staining with Coomassie methanol, 10% aceticstain acid for 80 min prior to

staining

Incompatible sample Consider minimizing

components salts, detergents, andsolvents in samplepreparation and samplebuffer

Bands are not present Proteins have Use a smaller poreor are missing from the transferred through the size membraneblotting membrane* membrane Decrease the transfer

time Decrease the voltage

*For more Western blot troubleshooting suggestions, see the Mini Trans-blot Electrophoretic Transfer Cell Instruction Manual

(1703930) or the Trans-BlotSD Semi-Dry Electrophoretic Transfer Cell Instruction Manual (1703940).

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Appendix AStock SolutionsBuffer Notes

SDS-PAGE running 10x Stock Running buffer should

buffer Tris base 15.0 g be ~pH 8.3. Do not adjustGlycine 72.0 g the pH

SDS 5.0 gTo 500 ml with DI H

2O

SDS-PAGE sample 2x Stock

buffer 0.5M Tris-HCl, pH 6.8 1.0 ml

10% (w/v) SDS 1.6 ml

Glycerol 2.0 ml

1.0% Bromophenol Blue 0.08 ml

2-Mercaptoethanol 0.4 ml

DI H2O 2.92 ml

Total Volume 8.0 ml

Native PAGE running 10x Stock Running buffer should

buffer Tris base 15.0 g be ~pH 8.3. Do not adjustGlycine 72.0 g the pH

To 500 ml with DI H2O

Native PAGE sample 2x Stock

buffer 0.5M Tris-HCl, pH 6.8 1.0 ml

Glycerol 2.2 ml

1% Bromophenol Blue 0.08 ml

DI H2O 3.72 ml

Total Volume 8.0 ml

0.5 M Tris-HCl Tris base 6.06 g Adjust to pH 6.8 with HCl.

DI H2O ~60 ml Make to 100 ml with

Total Volume 100 ml DI H2O. Store at 4C

10% SDS SDS 1.0 g Stir gently


1% Bromophenol Blue Bromophenol Blue 100 mg Stir gently


Coomassie Blue R-250 Methanol (40%) 400 ml Dissolve Coomassie R-250

staining solution (0.1%) Acetic Acid (10%) 100 ml in methanol/acetic acid.

Coomassie Blue R-250 (0.1%) 1.0 g Add DI H2O to a final

To 1,000 ml with DI H2O volume of 1,000 ml

Coomassie Blue R-250 Methanol 400 ml

destaining solution Acetic acid 100 ml

DI H2O 500 ml

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Appendix BTotal Protein Blot Stains

Appendix CRelated Literature

Name Bulletin Number

Mini-PROTEANTetra Cell Instruction Manual 10007296

Mini-PROTEAN 3 Instruction Manual 4006157

Mini-PROTEAN 3 Dodeca Cell Instruction Manual 4006191

Mini Trans-BlotInstruction Manual M1703930

Criterion Blotter Instruction Manual 4006190

Trans-BlotCell Instruction Manual 1703910

Trans-Blot SD Cell Quick Reference Guide 4006066

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

Blotting Membrane Brochure 1939

Western Blotting Detection Reagent Brochure 2032

Ready-to-Run Buffers and Solutions Brochure 2317

Little Book of Standards 2414

Model 583 Gel Dryer Instruction Manual M1651740

GelAir Drying System Instruction Manual 4006040

Method Sensitivity OptimalProtein

Load

Advantages Disadvantages Imaging

Number

SYPRORubyprotein blotstain

28 ng ~0.2g/band

Compatiblewith massspectrometry,Edman-basedsequencing,and standardimmunologicalprocedures

Multiple stepprotocol;requiresimaginginstrument formaximumsensitivity

Fluorescencevisualizationwith UV epi-illumination orlaser scanning

4006173

ColloidalGold stain

1 ng ~0.1g/band

Sensitive, onestep

Not compatiblewith nylonmembranes

Photographywith whitelight orreflectancedensitometry

LIT294

Amido Black10B

1001,000 ng ~5.0 g/band Standardmembrane,economical

Low sensitivitystain

Photographywith whitelight orreflectancedensitometry

9130

Instruction

Manual

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Appendix DOrdering Information

D.1 Mini-PROTEANTGX Precast Gels

10 Gels per box 2 Gels per box

10-Well 15-Well IPG Comb 10-Well

30 l/well 15 l/well 7 cm IPG Strip 30 l

7.5% 456-1023 456-1026 456-1021 456-1023S

10% 456-1033 456-1036 456-1031 456-1033S

12% 456-1043 456-1046 456-1041 456-1043S

415% 456-1083 456-1086 456-1081 456-1083S

420% 456-1093 456-1096 456-1091 456-1093S

Any kD 456-9033 456-9036 456-9031 456-9033S

D.2 Premixed Running and Sample BuffersCatalogNumber Product Description

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

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

161-0737 Laemmli Sample Buffer, 30 ml

161-0738 Native Sample Buffer, 30 ml

161-0734 10x Tris/Glycine, 1 L

161-0771 10x Tris/Glycine, 5 L cube

161-0778 10x Tris/CAPS, 1 L

161-0780 10x Phosphate Buffered Saline, 1 L

170-6435 10x Tris Buffered Saline, 1 L161-0783 1x Phosphate Buffered Saline With 1% Casein, 1 L

161-0782 1x Tris Buffered Saline With 1% Casein, 1 L

D.3 Individual Reagents

CatalogNumber Product Description

161-0719 Tris, 1 kg

161-0716 Tris, 500 g

161-0717 Glycine, 250 g

161-0718 Glycine, 1 kg

161-0724 Glycine, 2 kg

161-0301 SDS, 100 g161-0302 SDS, 1 kg

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

161-0418 SDS Solution, 20% (w/v), 1 L

170-6404 Blotting-Grade Blocker, 300 g

161-0710 2-Mercaptoethanol, 25 ml

161-0610 Dithiothreitol, 1 g

161-0611 Dithiothreitol, 5 g

161-0404 Bromophenol Blue, 10 g

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D.4 Total Protein Gel and Blot Stains


161-0786 Bio-Safe Coomassie Stain, 1 L

161-0400 Coomassie Brilliant Blue R-250, 10 g161-0436 Coomassie Blue R-250 Stain Solution, 1 L

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

161-0443 Silver Stain Kit

161-0449 Silver Stain Plus Kit

161-0481 Dodeca Silver Stain 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

170-3125 SYPRO Ruby Protein Gel Stain, 1 L

161-0490 Flamingo Fluorescent Gel Stain (10 x), 20 ml



161-0495 Oriole Fluorescent Protein Gel Stain (1 x), 200 ml

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

161-0497 Oriole Fluorescent Protein Gel Stain Kit, 5 L

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D.5 Immunoblot Detection

See related literature in Appendix C for information on Western blotting and gel drying.

D.6 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 g170-8238 Amplified Opti-4CN Substrate Kit

170-8235 Opti-4CN Substrate Kit

170-6432 AP Conjugate Substrate Kit

170-5012 Immun-Star Substrate Pack

Method Sensitivity OptimalProtein Load

Advantages Disadvantages Imaging

4CN colorimetric(HRP)

500 pg ~0.25g/band

Fast detection Results mayfade

Photography withwhite light or

reflectancedensitometry

DAB colorimetric(HRP)

500 pg ~0.25g/band

Fast detection Contains toxicchemicals

Photography withwhite light orreflectancedensitometry

Opti-4CNcolorimetric(HRP)

100 pg ~0.05g/band

Color doesnot fade

Moreexpensive than4CN


Amplified Opti-4CN colorimetric(HRP)

10 pg ~0.005g/band

Highsensitivity, lowbackground

Amplificationrequiresadditionalsteps


BCIP/NBT

colorimetric

100 pg ~0.05

g/band

Sensitive,

multipleantigen

May detect

endogenous(AP) enzymeactivity

Photography with

white light orreflectancedensitometry

Immun-Starchemiluminescent(AP)

10 pg

~0.005g/band

Long-lastingsignal, shortand multipleexposurespossible

Requiresvisualizationon film orinstrumentation

Chemiluminescentvisualization withfilm or imager

Immun-StarchemiluminescentHRP

13 pg

~0.005g/band

Intensifiessignal output,very sensitive

Requiresvisualization on

film orinstrumentation


Immun-StarWesternC (HRP)

10 fg

~0.005g/band Long-lastingsignal, shortand multipleexposurespossible

Requiresvisualzation onfilm orinstrumentation


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D.7 Blotting MembranesCatalogNumber Product Description

162-0232 0.2 m Nitrocellulose/Filter Paper Sandwich, 8.5 x 13.5 cm, 20 pack

162-0233 0.2 m Nitrocellulose/Filter Paper Sandwich, 8.5 x 13.5 cm, 50 pack162-0234 0.45 m Nitrocellulose/Filter Paper Sandwich, 8.5 x 13.5 cm, 20 pack

162-0235 0.45 m Nitrocellulose/Filter Paper Sandwich, 8.5 x 13.5 cm, 50 pack

162-0236 Sequi-Blot PVDF/Filter Paper Sandwich, 8.5 x 13.5 cm, 20 pack

162-0237 Sequi-Blot PVDF/Filter Paper Sandwich, 8.5 x 13.5 cm, 50 pack

D.8 Protein Standards


161-0363 Precision Plus Protein Unstained Standards (10250 kD), 1,500 l,

150 applications

161-0373 Precision Plus Protein All Blue Prestained Standards (10250 kD),

500 l, 50 applications161-0374 Precision Plus Protein Dual Color Standards (10250kD), 500 l,

50 applications

161-0375 Precision Plus Protein Kaleidoscope Standards (10250 kD), 500 l,

50 applications

161-0376 Precision Plus Protein WesternC Standards (10250kD), 250 l,

50 applications

161-0385 Precision Plus Protein WesternC Pack (10250kD), 50 applications

each of standard and StrepTactin-HRP

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

161-0320 2-D SDS-PAGE Standards, Unstained, 500 l

D.9 EquipmentCatalogNumber Product Description

165-8004 Mini-PROTEAN Tetra Cell

165-4100 Mini-PROTEAN 3 Dodeca Cell

170-3930 Mini Trans-BlotElectrophoretic Transfer Cell

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

164-5050 PowerPac Basic Power Supply

164-5052 PowerPac HC High-Current Power Supply

164-5070 PowerPac Universal Power Supply

164-5056 PowerPac HV Power Supply

165-1789 Hydrotech Gel Drying System, 100/120V

165-1790 Hydrotech Gel Drying System, 220/240V

165-1771 GelAir Drying System, 115V, 60Hz

165-1772 GelAir Drying System, 230V, 50Hz

SYPRO is a trademark of Molecular Probes, Inc. Bio-Rad is licensed to sell SYPRO products for researchuse only, under US Patent 5,616,502.

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Bio-RadLaboratories, Inc.

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