Mini-PROTEAN®

Precast Gels
Instruction 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)
 Table of Contents
Section 1 General Information ...................................................................1
1.1 Introduction .............................................................................................1
1.2 Mini-PROTEAN Precast Gel Specifications.............................................2
1.3 Important Notes ......................................................................................3
1.4 Mini-PROTEAN Comb Configurations.....................................................3
Section 2 Setup and Basic Operation ........................................................3
2.1 Required Materials ..................................................................................3
2.2 Mini-PROTEAN Precast Gel Set-Up Overview........................................4
2.3 Assembling the Mini-PROTEAN Tetra Cell Electrophoresis Module ........5
Section 3 SDS-PAGE ...................................................................................6
3.1 Introduction .............................................................................................6
3.2 Mini-PROTEAN TGX Gel Composition....................................................7
3.3 Mini-PROTEAN TGX Gel Selection Guide ..............................................7
3.4 SDS-PAGE Buffers .................................................................................8
3.5 Sample Preparation ................................................................................8
3.6 Running Conditions.................................................................................8
Section 4 Native PAGE ................................................................................8
4.1 Introduction .............................................................................................8
4.2 Native PAGE Buffers...............................................................................8
4.3 Sample Preparation ................................................................................8
4.4 Running Conditions.................................................................................8
Section 5 Buffers.........................................................................................9
Section 6 Total Protein Gel Stains for SDS-PAGE and Native PAGE
Detection ...................................................................................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 ................................................................15
D.1 Mini-PROTEAN TGX Precast Gels .................................................15
D.2 Premixed Running and Sample Buffers ..........................................15
D.3 Individual Reagents ........................................................................15
D.4 Total Protein Gel and Blot Stains ....................................................16
D.5 Immunoblot Detection.....................................................................17
D.6 Immunoblot Detection Reagents.....................................................17
D.7 Blotting Membranes........................................................................18
D.8 Protein Standards...........................................................................18
D.9 Equipment ......................................................................................18
Section 1
General Information
1.1 Introduction
Mini-PROTEAN® precast gels greatly simplify polyacrylamide gel electrophoresis. They are
specifically 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 stacking
layer. 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 proof
storage pouch with gel buffer containing 0.02% sodium azide.
The migration pattern of proteins on Mini-PROTEAN TGX™ precast gels is similar to that
observed 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 contain
no sodium dodecyl sulfate (SDS) and can therefore be used for either sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) or native gel electrophoresis depending upon
the 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 precast
gels 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 electrode
modules for tank blotting, 2-D electrophoresis and electroelution.
The Mini-PROTEAN 3 Dodeca cell is a multi-cell for high through-put system gel
electrophoresis. It can run up to 12 identical polyacrylamide gels simultaneously. The Dodeca
cell includes clamping frames, buffer dams, and a drain line.

1
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 2°C and 8°C; DO NOT FREEZE
Mini-PROTEAN Tetra Cell Specifications
Casting stand Polycarbonate
Pin, retaining ring and spring Stainless steel
Casting frames Polysulfone
Gray 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” diameter
Gasket, 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
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 different
run rates.
• When running 1 or 2 gels in the Tetra cell, use the electrode assembly (with the banana
plugs), 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 be
used.
• When running 1 or 2 gels only, DO NOT place the companion running module in the
tank. 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 2°C and 8°C. Avoid freezing or prolonged storage above
8°C. 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 of
the module. To prevent the gels from shifting during the locking step, firmly and evenly
grip 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 µl
15 well 15 µl
IPG 7 cm ReadyStrip™ IPG strip

Section 2
Setup and Basic Operation
2.1 Required Materials
• Clean Mini-PROTEAN® Tetra cell tank
• Electrophoresis module: to run 1 or 2 gels, use the electrode module. To run 3 or 4
gels, 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
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 wells
with 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 the
line on the bottom of the gel cassette (approximately 30–40 min). At the completion of the
run, disconnect the cell and remove the cassette.
5. Open Cassette: Align the arrow on the opening key with the arrows marked on the
cassette. 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 plates
beginning from the top of the cassette.
6. Remove Gel: Gently remove the gel from the cassette.

4
2a 2b 2c

Clamping
Frame
Gasket

Notch

Gel
Cassette
Short Plate
2d Long Plate 2f

Gel Support
2e

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; gel
supports are molded into the bottom of the electrode assembly. There are two supports
on 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 the
first 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 of
the frame (see Figure 2b). It is critical that gel cassettes be placed into the electrode
assembly 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 of
gels (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 the
green 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 in
place), slide the green arms of the clamping frame over the gels, locking them into
place (see Figure 2c).

5
5. The wing clamps of the electrode assembly lift each gel cassette up against the notch
in the green gasket, forming a seal (Figure 2d). Check again to make certain that the
short 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 steps
2a–2d with the companion running module.

Section 3
SDS-PAGE
3.1 Introduction
Mini-PROTEAN® TGX™ precast gels provide a versatile system for sodium dodecyl
sulfate 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. Proteins
have an intermediate mobility, causing them to concentrate, or stack, into a narrow zone at
the 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. The
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 reductant, usually
2-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 bonds
between 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 distance
depends primarily on molecular weight. Molecular mass is determined by plotting the
logarithm of protein molecular mass vs. the relative mobility (Rf) of the protein (Rf = 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-PAGE
separations are obtained in gels lacking SDS, provided that the sample buffer and running
buffer contain sufficient SDS to maintain SDS saturation during electrophoresis. The
recommended 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 may
also be used for native electrophoresis (see Section 4).
Mini-PROTEAN TGX precast gels differ from the standard Laemmli system (Tris-HCl
SDS-PAGE) gels due to a proprietary modification to their formulations that provides the
gels 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 or
reagents are required.

6
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 mm
from 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* Run
Type Separation Range Buffer Buffer Voltage/Current** Time***

TGX 7.5 40–200 kD SDS-PAGE SDS-PAGE 200 V constant 38 min

sample buffer running Starting current
buffer (per gel): 37 mA
Final current
(per gel): 23 mA

TGX 10 30–150 kD SDS-PAGE SDS-PAGE 200 V constant 38 min

sample running Starting current
buffer buffer (per gel): 37 mA
Final current
(per gel): 23 mA

TGX 12 20–120 kD SDS-PAGE SDS-PAGE 200 V constant 38 min

sample running Starting current
buffer buffer (per gel): 37 mA
Final current
(per gel): 23 mA

TGX 4–15 20–250 kD SDS-PAGE SDS-PAGE 200 V constant 30 min

sample running Starting current
buffer buffer (per gel): 50 mA
Final current
(per gel): 33 mA

TGX 4–20 10–200 kD SDS-PAGE SDS-PAGE 200 V constant 30 min

sample running Starting current
buffer buffer (per gel): 50 mA
Final current
(per gel): 33 mA

TGX Any kD****10–200 kD SDS-PAGE SDS-PAGE 200 V constant 28 min

sample running Starting current
buffer buffer (per gel): 50 mA
Final 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
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 detection
method used. (See Section 6 for approximate stain sensitivities). Add 50 µl 2-mercaptoethanol
per 950 µl 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). Dilute
1 part sample with at least 1 part sample buffer with added reductant. Heat the mixture at
95°C 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 depending
on the gel type (see Section 3.3).

Section 4
Native PAGE
4.1 Introduction
Mini-PROTEAN® TGX™ gels are made without SDS, allowing native separations using
SDS- and reductant-free sample and running buffers. The nonreducing and nondenaturing
environment 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 and
shape of the protein as well as its molecular weight and net charge. Native PAGE is therefore
not 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 can
be 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 basic
proteins (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
Section 5
Buffers (see Appendix A for Stock Solutions)
Name Working Concentration Notes Pre-Mixed Alternative
SDS-Page 1X Running buffer should 10x Tris/Glycine/SDS, 1 L,
running buffer 25 mM Tris - base be ~ pH 8.3. Do not 161-0732
192 mM glycine adjust the pH 10x Tris/Glycine/SDS, 5 L cube,
0.1% (w/v) SDS 161-0772

SDS-PAGE 2X Dilute 1 part sample Laemmli sample buffer, 30 ml,

sample buffer 62.5 mM Tris -HCl, pH 6.8 with 1 part sample 161-0737
2% (w/v) SDS buffer. More sample
25% (v/v) glycerol buffer can be added if
0.01% (w/v) Bromophenol Blue necessary. 1 part
5% (v/v) 2-mercaptoethanol or sample to 2 parts
350 mM DTT (added fresh) sample buffer dilution
also works. Dry
samples can be
dissolved directly into
the sample buffer
Native PAGE 25 mM Tris -Base Running buffer should 10x Tris/Glycine, 1 L,
running buffer 192 mM glycine be ~ pH 8.3. Do not 161-0734
working adjust the pH 10x Tris/Glycine, 5 L cube,
concentration 161-0771
Native PAGE 62.5 mM Tris-HCl, pH 6.8 Dilute 1 part sample Native sample buffer, 30 ml,
sample buffer 40% glycerol with 1 part sample 161-0738
0.01% Bromophenol Blue buffer. More sample
buffer can be added if
necessary. 1 part
sample to 2 parts
sample buffer dilution
also works. Dry
samples can be
dissolved directly into
the sample buffer

9
Section 6
Total Protein Gel Stains for SDS-PAGE and Native
PAGE Detection
Method Sensitivity Optimal Advantages Disadvantages Imaging Instruction
Protein Manual
Load Number
Coomassie 36–47 ng ~0.5 Laboratory Requires MeOH Photography Consult
Blue R-250 µg/band standard with white light or scientific
transmission literature
densitometry
(Gel Doc™ or
GS-800™)
Bio-Safe™ 8–28 ng ~0.5 Nonhazardous Photography 4307051
Coomassie µg/band with white light or
stain transmission
densitometry
(Gel Doc or
GS-800)
Zinc stain 6–12 ng ~0.2 High-contrast, Negative stain, Photography 4006082
µg/band fast, reversible must be with white light or
stain photographed, transmission
SDS-PAGE densitometry
only (Gel Doc or GS-
800)
Silver 0.6–1.2 ng ~0.01 Simple, Does not stain Photography LIT442
Stain™ Plus µg/band robust, mass Glycoproteins with white light or
kit spectrometry well transmission
compatible densitometry
(Gel Doc or
GS-800)
Silver stain 0.6–1.2 ng ~0.01 Stains Not mass Photography LIT34
µg/band complex spectrometry with white light or
proteins, i.e., compatible transmission
glycoproteins, densitometry
and (Gel Doc or
lipoproteins GS-800)
Dodeca™ 0.5–1.2 ng ~0.1 Convenient Photography 4110150
Silver Stain µg/band staining for a with white light or
Kit large number transmission
of gels densitometry
(Gel Doc or GS-
800)
SYPRO™ 1–10 ng ~0.2 Broad Requires Fluorescence 4006173
Ruby protein µg/band dynamic imaging visualization with
gel stain range, simple instrument for UV trans-
robust protocol maximum illumination or
sensitivity laser scanning
Flamingo™ 0.25–0.5 ng ~0.01 Broad Requires Fluorescence 10003321
Fluorescent µg/band dynamic imaging visualization
range, mass instrument for with UV trans-
spec maximum illumination or
compatible sensitivity laser scanning
(best option)
Oriole 0.5 ng ~0.2 High Will not work Fluorescence 10017295
Fluorescent µg/band sensitivity with visible visualization with
protein gel Broad excitation UV transil-
stain dynamic range lumination (Gel
Doc, Chemi Doc)

10
Section 7
Troubleshooting
Problem
Current is zero or less than
expected and samples do not
migrate into gel
Bands “smile” across gel,
band pattern curves upward
at both sides of the gel
Smiling or frowning bands
within the gel lane
Skewed or distorted bands,
lateral band spreading
Vertical streaking
Cause
• Tape at the bottom of
the cassette not
removed
• Insufficient buffer in inner
buffer chamber
• Insufficient buffer
in outer buffer
chamber
• Electrical disconnection
• Excess heating of gel
• Insufficient buffer
• Overloaded proteins
• Sample
preparation/buffer
issues
• Running speed
• Excess salt in samples
• Insufficient sample
buffer or wrong
formulation
• Overloaded samples
Solution
• Remove tape
• Fill buffer chamber with
700 ml running buffer
• Make sure the inner
and outer buffer
chambers are
sufficiently filled to
ensure that the wells of
the gel are completely
covered
• Check electrodes and
connections
• Check buffer composition
• Do not exceed
recommended running
conditions
• Make sure the inner
and outer buffer
chambers are
sufficiently filled to
ensure that the wells of
the gel are completely
covered
• Load less protein
• Consider minimizing
salts, detergents and
solvents in sample
preparation and sample
buffer
• Check to make sure the
correct voltage has
been set
• Remove salts from
sample by dialysis or
desalting column prior
to sample preparation
• Check buffer
composition and
dilution instructions
• Dilute sample

11
Problem Cause Solution
Vertical streaking • Sample precipitation • Selectively remove
predominant protein in
the sample
• Dilute sample in more
sample buffer
• Insoluble materials • Centrifuge samples to
in the samples remove particulates
(e.g., membranes) prior to sample loading
Gels run faster than • Running buffer is too • Check buffer
expected concentrated and gel composition
temperature is too high
• Incorrect running buffer
type is used
Artifact bands at ~60–70 kD • Possible skin keratin • Thoroughly clean all
contamination dishware and wear
gloves while handling
and loading gel
• Filter all solutions
through 0.2 µm or
0.45 µm filter
Leaking from inner • Incomplete gasket seal • Wet the gasket with
buffer chamber running buffer before
use
• Improper assembly of • Check that the top edge
the gel into the of the short plate fits
electrode/companion under the notch at the
assembly top of the gasket
• Make sure that the top
of the short plate is
touching the green
gasket
Poor resolution • High sample volume • If possible, load a more
or fuzzy bands concentrated sample
in a lower volume of
sample buffer
• Diffuse sample loading • Load sample with
zone syringe or gel loading
pipette tips
• Sample diffusion during • Fix gel with 40%
staining with Coomassie methanol, 10% acetic
stain acid for 80 min prior to
staining
• Incompatible sample • Consider minimizing
components salts, detergents, and
solvents in sample
preparation and sample
buffer
Bands are not present • Proteins have • Use a smaller pore
or are missing from the transferred through the size membrane
blotting 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-Blot® SD Semi-Dry Electrophoretic Transfer Cell Instruction Manual (1703940).

12
Appendix A
Stock Solutions
Buffer Notes
SDS-PAGE running 10x Stock Running buffer should
buffer Tris base 15.0 g be ~pH 8.3. Do not adjust
Glycine 72.0 g the pH
SDS 5.0 g
To 500 ml with DI H2O
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 adjust
Glycine 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 4°C

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
Appendix B
Total Protein Blot Stains
Method Sensitivity Optimal Advantages Disadvantages Imaging Instruction
Protein Manual
Load Number
SYPRO 2–8 ng ~0.2 Compatible Multiple step Fluorescence 4006173
Ruby µg/band with mass protocol; visualization
protein blot spectrometry, requires with UV epi-
stain Edman-based imaging illumination or
sequencing, instrument for laser scanning
and standard maximum
immunological sensitivity
procedures
Colloidal 1 ng ~0.1 Sensitive, one Not compatible Photography LIT294
Gold stain µg/band step with nylon with white
membranes light or
reflectance
densitometry
Amido Black 100–1,000 ng ~5.0 µg/band Standard Low sensitivity Photography 9130
10B membrane, stain with white
economical light or
reflectance
densitometry

Appendix C
Related Literature
Name Bulletin Number

Mini-PROTEAN® Tetra Cell Instruction Manual 10007296

Mini-PROTEAN 3 Instruction Manual 4006157
Mini-PROTEAN 3 Dodeca Cell Instruction Manual 4006191
Mini Trans-Blot® Instruction Manual M1703930
Criterion™ Blotter Instruction Manual 4006190
®
Trans-Blot Cell 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

14
Appendix D
Ordering Information
D.1 Mini-PROTEAN® TGX™ Precast Gels

10 Gels per box 2 Gels per box

10-Well 15-Well IPG Comb 10-Well
30 µl/well 15 µl/well 7 cm IPG Strip 30 µl

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
4–15% 456-1083 456-1086 456-1081 456-1083S
4–20% 456-1093 456-1096 456-1091 456-1093S
Any kD™ 456-9033 456-9036 456-9031 456-9033S

D.2 Premixed Running and Sample Buffers

Catalog
Number 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 L
161-0783 1x Phosphate Buffered Saline With 1% Casein, 1 L
161-0782 1x Tris Buffered Saline With 1% Casein, 1 L
D.3 Individual Reagents
Catalog
Number 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 g
161-0302 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
D.4 Total Protein Gel and Blot Stains
Catalog
Number Product Description
161-0786 Bio-Safe Coomassie Stain, 1 L
161-0400 Coomassie Brilliant Blue R-250, 10 g
161-0436 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
D.5 Immunoblot Detection
Method Sensitivity Optimal Advantages Disadvantages Imaging
Protein Load
4CN colorimetric 500 pg ~0.25 Fast detection Results may Photography with
(HRP) µg/band fade white light or
reflectance
densitometry
DAB colorimetric 500 pg ~0.25 Fast detection Contains toxic Photography with
(HRP) µg/band chemicals white light or
reflectance
densitometry
Opti-4CN™ 100 pg ~0.05 Color does More Photography with
colorimetric µg/band not fade expensive than white light or
(HRP) 4CN reflectance
densitometry
Amplified Opti- 10 pg ~0.005 High Amplification Photography with
4CN colorimetric µg/band sensitivity, low requires white light or
(HRP) background additional reflectance
steps densitometry

BCIP/NBT 100 pg ~0.05 Sensitive, May detect Photography with

colorimetric µg/band multiple endogenous white light or
antigen (AP) enzyme reflectance
activity densitometry

Immun-Star™ 10 pg ~0.005 Long-lasting Requires Chemiluminescent

chemiluminescent µg/band signal, short visualization visualization with
(AP) and multiple on film or film or imager
exposures instrumentation
possible

Immun-Star 1–3 pg ~0.005 Intensifies Requires Chemiluminescent

chemiluminescent µg/band signal output, visualization on visualization with
HRP very sensitive film or film or imager
instrumentation

Immun-Star 10 fg ~0.005 Long-lasting Requires Chemiluminescent

WesternC™ (HRP) µg/band signal, short visualzation on visualization with
and multiple film or film or imager
exposures instrumentation
possible

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

D.6 Immunoblot Detection Reagents

Catalog
Number 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 g
170-8238 Amplified Opti-4CN Substrate Kit
170-8235 Opti-4CN Substrate Kit
170-6432 AP Conjugate Substrate Kit
170-5012 Immun-Star Substrate Pack

17
D.7 Blotting Membranes
Catalog
Number Product Description
162-0232 0.2 µm Nitrocellulose/Filter Paper Sandwich, 8.5 x 13.5 cm, 20 pack
162-0233 0.2 µm Nitrocellulose/Filter Paper Sandwich, 8.5 x 13.5 cm, 50 pack
162-0234 0.45 µm Nitrocellulose/Filter Paper Sandwich, 8.5 x 13.5 cm, 20 pack
162-0235 0.45 µm 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

Catalog
Number Product Description
161-0363 Precision Plus Protein™ Unstained Standards (10–250 kD), 1,500 µl,
150 applications
161-0373 Precision Plus Protein All Blue Prestained Standards (10–250 kD),
500 µl, 50 applications
161-0374 Precision Plus Protein Dual Color Standards (10–250kD), 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 (10–250kD), 250 µl,
50 applications
161-0385 Precision Plus Protein WesternC Pack (10–250kD), 50 applications
each of standard and StrepTactin-HRP
161-0317 SDS-PAGE Standards, broad range, 200 µl
161-0320 2-D SDS-PAGE Standards, Unstained, 500 µl

D.9 Equipment
Catalog
Number Product Description
165-8004 Mini-PROTEAN Tetra Cell
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
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 research
use only, under US Patent 5,616,502.

18
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