tgx gels instructions
TRANSCRIPT
-
8/14/2019 TGX Gels Instructions
1/24
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)
-
8/14/2019 TGX Gels Instructions
2/24
-
8/14/2019 TGX Gels Instructions
3/24
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
-
8/14/2019 TGX Gels Instructions
4/24
-
8/14/2019 TGX Gels Instructions
5/24
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.
1
-
8/14/2019 TGX Gels Instructions
6/24
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)
2
-
8/14/2019 TGX Gels Instructions
7/24
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
3
-
8/14/2019 TGX Gels Instructions
8/24
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.
4
-
8/14/2019 TGX Gels Instructions
9/24
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
-
8/14/2019 TGX Gels Instructions
10/24
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.
6
-
8/14/2019 TGX Gels Instructions
11/24
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
TGX 12 20120 kD SDS-PAGE SDS-PAGE 200 V constant 38 minsample running Starting currentbuffer buffer (per gel): 37 mA
Final current
(per gel): 23 mA
TGX 415 20250 kD SDS-PAGE SDS-PAGE 200 V constant 30 minsample running Starting currentbuffer buffer (per gel): 50 mA
Final current(per gel): 33 mA
TGX 420 10200 kD SDS-PAGE SDS-PAGE 200 V constant 30 minsample running Starting currentbuffer buffer (per gel): 50 mA
Final current(per gel): 33 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.
7
-
8/14/2019 TGX Gels Instructions
12/24
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.
8
-
8/14/2019 TGX Gels Instructions
13/24
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
9
-
8/14/2019 TGX Gels Instructions
14/24
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
10
-
8/14/2019 TGX Gels Instructions
15/24
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
11
-
8/14/2019 TGX Gels Instructions
16/24
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).
12
-
8/14/2019 TGX Gels Instructions
17/24
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
To 10 ml with DI H2O
1% Bromophenol Blue Bromophenol Blue 100 mg Stir gently
To 10 ml with DI H2O
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
13
-
8/14/2019 TGX Gels Instructions
18/24
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
14
-
8/14/2019 TGX Gels Instructions
19/24
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
15
-
8/14/2019 TGX Gels Instructions
20/24
D.4 Total Protein Gel and Blot Stains
CatalogNumber Product Description
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
161-0491 Flamingo Fluorescent Gel Stain (10 x), 100 ml
161-0492 Flamingo Fluorescent Gel Stain (10 x), 500 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
16
-
8/14/2019 TGX Gels Instructions
21/24
D.5 Immunoblot Detection
See related literature in Appendix C for information on Western blotting and gel drying.
D.6 Immunoblot Detection Reagents
CatalogNumber Product Description
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
Photography withwhite light orreflectancedensitometry
Amplified Opti-4CN colorimetric(HRP)
10 pg ~0.005g/band
Highsensitivity, lowbackground
Amplificationrequiresadditionalsteps
Photography withwhite light orreflectancedensitometry
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
Chemiluminescentvisualization withfilm or imager
Immun-StarWesternC (HRP)
10 fg
~0.005g/band Long-lastingsignal, shortand multipleexposurespossible
Requiresvisualzation onfilm orinstrumentation
Chemiluminescentvisualization withfilm or imager
17
-
8/14/2019 TGX Gels Instructions
22/24
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
CatalogNumber Product Description
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.
18
-
8/14/2019 TGX Gels Instructions
23/24
-
8/14/2019 TGX Gels Instructions
24/24
Life ScienceGroup
Bio-RadLaboratories, Inc.
Web site www.bio-rad.com USA 800 4BIORAD Australia 61 02 9914 2800 Austria 01 877 89 01 Belgium 09 385 55 11 Brazil55 21 3237 9400Canada 905 364 3435 China 86 21 6426 0808 Czech Republic420 241 430 532 Denmark44 52 10 00 Finland09 804 22 00 France 01 47 95 69 65Germany089 318 84 0 Greece 30 210 777 4396 Hong Kong 852 2789 3300 Hungary36 1 455 8800 India 91 124 4029300 Israel03 963 6050Italy39 02 216091 Japan 03 6361 7000 Korea 82 2 3473 4460 Mexico 52 555 488 7670 The Netherlands 0318 540666 New Zealand0508 805 500Norway23 38 41 30 Poland48 22 331 99 99 Portugal351 21 472 7700 Russia 7 495 721 14 04 Singapore 65 6415 3188 South Africa 27 861 246 723Spain 34 91 590 5200 Sweden 08 555 12700 Switzerland061 717 95 55 Taiwan 886 2 2578 7189 United Kingdom 020 8328 2000