Bio-Rad Mini-PROTEAN TGX Precast Gels for 2-D Electrophoresis User Manual
Mini-PROTEAN® Precast Gels
Instruction Manual and Application Guide
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Contents
Chapter 1: Mini-PROTEAN® Precast Gels ............................................. 1
1.1 Introduction ................................................................... 1
1.2 Gel Formulations ............................................................... 2
1.3 Comb Configurations ........................................................... 2
1.4 Specifications ................................................................. 2
1.5 Storage Conditions ............................................................ 3
1.6 Important Notes ............................................................... 3
Chapter 2: Setup and Basic Operation ............................................... 4
2.1 Workflow Overview ............................................................. 4
2.2 Required Materials ............................................................. 5
2.3 Setting Up and Running Mini-PROTEAN Gels in the Mini-PROTEAN® Tetra Cell............... 5
2.4 Removing the Gel .............................................................. 7
Chapter 3: SDS-PAGE............................................................. 8
3.1 Introduction ................................................................... 8
3.2 Mini-PROTEAN® TGX™ and Mini-PROTEAN® TGX Stain-Free™ Gels ....................... 8
3.3 SDS-PAGE Buffers ............................................................ 10
3.4 Sample Preparation............................................................ 10
3.5 Running Conditions ............................................................ 10
Chapter 4: Native PAGE .......................................................... 12
4.1 Introduction .................................................................. 12
4.2 Mini-PROTEAN TGX and Mini-PROTEAN TGX Stain-Free Gels........................... 12
4.3 Native PAGE Buffers .......................................................... 13
4.4 Sample Preparation............................................................ 13
4.5 Running Conditions ............................................................ 13
Chapter 5: Stain-Free System ..................................................... 14
5.1 Introduction .................................................................. 14
5.2 Stain-Free Workflow ........................................................... 15
5.3 Electrophoresis with Mini-PROTEAN TGX Stain-Free Gels .............................. 15
5.4 Stain-Free Detection ........................................................... 15
Chapter 6: Peptide Analysis ....................................................... 16
6.1 Introduction .................................................................. 16
6.2 Mini-PROTEAN Tris-Tricine Gels ................................................. 16
6.2.1 Gel Composition .......................................................... 16
6.2.2 Gel Selection Guide........................................................ 16
6.3 Peptide Analysis Buffers ........................................................ 17
6.4 Sample Preparation............................................................ 17
6.5 Running Conditions ............................................................ 17
Chapter 7: Nondenaturing Nucleic Acid PAGE ........................................ 18
7.1 Introduction .................................................................. 18
7.2 Mini-PROTEAN TBE Gels ....................................................... 18
7.2.1 Gel Composition .......................................................... 18
7.2.2 Gel Selection Guide........................................................ 18
7.3 Nondenaturing Nucleic Acid PAGE Buffers .......................................... 19
7.4 Sample Preparation............................................................ 19
7.5 Running Conditions ............................................................ 19
Chapter 8: Denaturing Nucleic Acid PAGE ........................................... 20
8.1 Introduction .................................................................. 20
8.2 Mini-PROTEAN TBE-Urea Gels ................................................... 20
8.2.1 Gel Composition .......................................................... 20
8.2.2 Gel Selection Guide........................................................ 20
8.3 Denaturing Nucleic Acid PAGE Buffers ............................................. 21
8.4 Sample Preparation............................................................ 21
8.5 Running Conditions ............................................................ 21
Chapter 9: 2-D Electrophoresis .................................................... 22
9.1 Introduction .................................................................. 22
9.2 Equilibration.................................................................. 22
9.3 Agarose Overlay .............................................................. 22
9.4 Second-Dimension Electrophoresis................................................ 22
Chapter 10: Detection............................................................ 23
10.1 SDS-PAGE and Native PAGE Detection ........................................... 23
10.2 Peptide Gel Staining .......................................................... 24
10.3 TBE Gel Staining ............................................................ 24
10.4 TBE-Urea Gel Staining ....................................................... 24
Chapter 11: Blotting ............................................................. 25
11.1 Introduction ................................................................. 25
11.2 Transfer ................................................................... 25
11.2.1 Transfer Buffers .......................................................... 25
11.2.2 Wet Transfer Using the Mini Trans-Blot® Module................................. 25
11.2.3 Transfer Using the Trans-Blot® Turbo™ System.................................. 26
11.2.4 Semi-Dry Transfer Using the Trans-Blot® SD Cell ................................ 27
11.3 Total Protein Blot Stains ....................................................... 28
11.4 Immunodetection ............................................................ 28
Chapter 12: Troubleshooting ...................................................... 29
Appendix A: Quick Start Guides.................................................... 31
SDS-PAGE (Mini-PROTEAN TGX Gels) ............................................. 32
Native PAGE (Mini-PROTEAN TGX Gels) ............................................ 33
Peptide Analysis (Mini-PROTEAN Tris-Tricine Gels) .................................... 34
Nondenaturing Nucleic Acid PAGE (Mini-PROTEAN TBE Gels)............................ 35
Denaturing Nucleic Acid PAGE (Mini-PROTEAN TBE-Urea Gels) .......................... 36
Appendix B: Buffers ............................................................. 37
Appendix C: Related Literature .................................................... 40
Appendix D: Ordering Information .................................................. 41
®
Mini-PROTEAN
1
Precast Gels
1.1 Introduction
Mini-PROTEAN precast gels are 7.2 cm x 8.6 cm gels designed for performing polyacrylamide gel
electrophoresis (PAGE) with the Mini-PROTEAN family of vertical electrophoresis cells, which includes
the Mini-PROTEAN® Tetra and Mini-PROTEAN® 3 Dodeca™ cells and the discontinued Mini-PROTEAN II
and Mini-PROTEAN 3 cells. The Mini Trans-Blot®, Trans-Blot® Turb o™, and Trans-Blot® SD blotting cells
and precut membrane sandwiches are also available for blotting applications with these gels.
Features of Mini-PROTEAN precast gels include:
n
Outlined and numbered well that simplify sample loading and identification
n
Capacity for up to 15 samples per gel
n
Bottom-open cassette design for easy gel handling and blotting setup
n
Easy-to-open cassette for faster downstream processing
n
Reference line at the bottom of the cassette indicates where the run should stop
(for optimum resolution across the separation range)
n
Excellent staining quality and transfer efficiency
n
No gel foot to remove prior to blotting
n
Mini-PROTEAN® TGX Stain-Free™ formulations, which enable rapid 5 min gel imaging
without staining and destaining
Comb (available in a range of options)
Numbered well outlines
Reference line for monitoring
progress of the run
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Mini-PROTEAN Precast Gels
1.2 Gel Formulations
Mini-PROTEAN precast gels are composed of polyacrylamide with a bisacrylamide crosslinker, and they
are available in a range of formulations (Table 1.1) and in a selection of single percentages and gradients.
Table 1.1. Mini-PROTEAN precast gel formulations.
Application Gel Formulation Sample Buffer Running Buffer
SDS-PAGE Mini-PROTEAN TGX™ Laemmli Tris/glycine/SDS
Mini-PROTEAN TGX Stain-Free
Native PAGE Mini-PROTEAN TGX Native Tris/glycine
Mini-PROTEAN TGX Stain-Free
Peptide analysis Mini-PROTEAN Tris-Tricine Tricine Tris/Tricine/SDS
dsDNA separation Mini-PROTEAN TBE Nucleic acid Tris/boric acid/EDTA (TBE)
ssDNA and RNA Mini-PROTEAN TBE-urea TBE-urea TBE
separation
1.3 Comb Configurations
Comb Type Well Volume
10-well 50 μl
10-well 30 μl
12-well 20 μl
15-we ll 15 μl
8 + 1 well* 30 μl
IPG/prep 7 cm ReadyStrip™ IPG strip (450 μl)
1.4 Specifications
Gel material Polyacrylamide
Gel dimensions 7.2 x 8.6 cm
Gel thickness 1.0 mm
Resolving gel height 6.2 cm (5.6 cm for 50 μl well)
Cassette dimensions 8.5 x 10 cm
Cassette material Styrene copolymer
Comb material Polycarbonate
Running buffer 750 ml for 1–2 gels, 1,000 ml for 3–4 gels (Mini-PROTEAN Tetra cell)
325 ml for 1–2 gels (Mini-PROTEAN II or Mini-PROTEAN 3 cell)
*
Multichannel pipet compatible.
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Instruction Manual and Application Guide
1.5 Storage Conditions
Table 1.2. Storage conditions for Mini-PROTEAN precast gels. Store gels flat. Shelf life is from date of manufacture;
expiration dates are printed on the packaging.
Storage
Temperature Gel Formulation Shelf Life
2–8°C Mini-PROTEAN TGX 12 months
Mini-PROTEAN TGX Stain-Free 12 months
Mini-PROTEAN Tris-Tricine 12 weeks
Mini-PROTEAN TBE 12 weeks
Mini-PROTEAN TBE-urea 8 weeks
1.6 Important Notes
Use each Mini-PROTEAN precast gel as soon as possible after removing it from the storage pouch.
Improper storage of Mini-PROTEAN precast gels can produce artifacts. Store gels flat and at 2–8°C.
Avoid freezing or prolonged storage above 8°C. If your gels have been stored improperly, discard them.
Do not run more than one gel type in the same apparatus at the same time. Different gel percentages
and formulations have different conductivities and different run times.
With the Mini-PROTEAN Tetra cell:
n
When running 1–2 gels:
Use the electrode assembly (with banana plugs), not the companion running module
(without banana plugs)
Do not place the companion running module in the tank. Doing so generates
excessive heat and degrades the quality of the electrophoretic separation
n
When running 3–4 gels, use both the electrode assembly and companion running module
n
When using voltages >200 V, fill the outer buffer chamber to the 4 gel (800 ml) mark
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Setup and Basic
2
Operation
2.1 Workflow Overview
Prepare sample and running buffers
Assemble Electrophoresis Cell
Prepare and Load Samples
Prepare Buffers
Prepare Gels and
Dilute in sample buffer
Perform Electrophoresis
SDS-PAGE (Chapter 3)
Native PAGE (Chapter 4)
Peptide Analysis (Chapter 6)
Nondenaturing Nucleic Acid PAGE (Chapter 7)
Denaturing Nucleic Acid PAGE (Chapter 8)
2-D Electrophoresis (Chapter 9)
Analyze the Separation
Blot the Gels (Optional)
(Chapter 10)
(Chapter 11)
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Instruction Manual and Application Guide
2.2 Required Materials
n
Mini-PROTEAN® precast gels
n
Mini-PROTEAN® Tetra cell (or Mini-PROTEAN® 3 Dodeca™, Mini-PROTEAN II or
Mini-PROTEAN 3 cell)
n
PowerPac™ Basic or PowerPac HC power supply (or equivalent); PowerPac HV or
PowerPac Universal required for high-voltage applications (>300 V)
n
Sample buffer
n
Running buffer (750 ml for 1–2 gels; 1,000 ml for 3–4 gels or when running at
voltages >200 V)
n
Opening lever (catalog #456-0000)
2.3 Setting Up and Running Mini-PROTEAN Gels in the
Mini-PROTEAN Tetra Cell
1. Remove the gels from the storage pouch and prepare them for assembly:
a. Remove the comb: Position thumb on the indentation (middle of comb) and remove the comb
by pulling upward in one smooth motion.
b. Remove the tape: Pull gently to remove the green tape from the bottom of the cassette.
If necessary, use the opening key or comb to help remove the tape at the corners.
c. Rinse the wells: Use a syringe, wash bottle, or disposable transfer pipet to rinse the wells with
Remove the comb
Remove the tape
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Mini-PROTEAN Precast Gels
running buffer. Straighten the sides of the
wells, if necessary.
2. Set the electrode assembly to the open
position on a clean, flat surface (A).
3. Place the gel cassettes into the electrode
assembly. Two cassettes are required to create
a functioning assembly; when using 1 or 3
gels, use the buffer dam (included with the cell)
to complete the assembly.
a. Place the first cassette with the short plate
facing inward and so the gel rests at a 30°
angle away from the center of the electrode
assembly. Make sure the electrode assembly
remains balanced and does not tip over.
b. Place the second gel or buffer dam on the
other side of the electrode assembly, again
by resting the gel on the supports. The gels
rest at 30° angles, one on either side of the
electrode assembly, tilting away from the
center of the frame (B).
4. Gently push both gels toward each other,
making sure that they rest firmly and squarely
against the green gasket that is built into the
electrode assembly. Align the short plates to
ensure the edge sits just below the notch at
the top of the green gasket (C).
5. While gently squeezing the gel cassettes
(or cassette and buffer dam) against the green
gaskets (maintaining constant pressure and
with both gels in place), slide the green arms
of the clamping frame one at a time over the
gels, locking them into place (D,E).
Clamping frame
Gasket
Notch
Gel cassette
Short plate
Long plate
Gel support
A
B
C
6. The wing clamps of the electrode assembly lift
each gel cassette up against the notch in the
green gasket, forming a seal. Check again that
D
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E
Instruction Manual and Application Guide
F
the short plates sit just below the notch at the top of
the green gasket (C).
If running more than 2 gels, repeat steps 2–6 with the
companion running module.
7. Place the electrophoresis module into the tank (F) and
fill the buffer chambers with 1x running buffer:
n
200 ml in the inner buffer chamber
n
550 ml (1–2 gels) or 800 ml (3–4 gels, or
>200 V) in the outer buffer chamber
8. Wash the sample wells with running buffer (if this was
not done earlier).
9. Load samples and run the gels using the running
conditions appropriate to your application. Stop the run when the dye front reaches the reference
line imprinted on the bottoms of the cassettes.
2.4 Removing the Gel
1. After electrophoresis is complete, turn off the power supply and disconnect the electrical leads.
2. Remove the lid from the tank and remove the gels from the cell. Pour off and discard the running
buf fer.
3. To open the cassette, align the arrow on the opening lever with the arrows marked on the cassette
and insert the lever between the cassette plates at indicated locations. Apply downward pressure to
break each seal. Do not twist the lever.
4. Pull the two plates apart from the top of the cassette, and gently remove the gel.
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3
SDS-PAGE
3.1 Introduction
Mini-PROTEAN® TGX™ (Tris-Glycine eXtended shelf life) gels provide a versatile system for separating
proteins by either molecular weight (SDS-PAGE) or mass-to-charge ratio (native PAGE). (See Chapter
4 for native PAGE applications and protocols.) This versatility is possible because the gels are made
without SDS; this allows the sample buffer and running buffer to determine the separation mechanism.
SDS-PAGE relies on a discontinuous buffer system. Two ions differing in electrophoretic mobility
(glycinate and chloride) form a moving boundary when voltage is applied. Proteins have an
intermediate mobility that causes them to concentrate, or stack, into a narrow zone at the beginning
of electrophoresis. As that zone moves through the gel, the sieving effect of the polyacrylamide gel
matrix causes proteins of different molecular weighs to move at different rates. This stacking effect is
responsible for the high resolving power of SDS-PAGE: the sample is loaded in a relatively broad zone,
and the moving boundary concentrates the proteins into sharp bands prior to separation.
Protein samples for SDS-PAGE are prepared using SDS and a thiol reducing agent, usually
β-mercaptoethanol or dithiothreitol (DTT). SDS forms complexes with proteins, giving them a rodlike
shape and similar mass-to-charge ratio. The reducing agent disrupts disulfide bonds between and
within proteins, allowing complete denaturation and dissociation. Heat treatment in the presence of
SDS and reducing agent effectively eliminates the effects of native charge and higher order structure on
electrophoretic mobility, so the migration distance depends primarily on molecular weight.
Molecular weight is estimated by plotting the logarithm of protein molecular weight vs. the relative
mobility (Rf) of the protein (Rf = distance migrated by the protein/distance migrated by the dye front)
or by using the point-to-point semilog interpolation method in Quantity One® or Image Lab™ software.
Refer to bulletins 3133, 3144, and 10014472 for more information.
3.2 Mini-PROTEAN TGX and
Mini-PROTEAN® TGX Stain-Free™ Gels
Mini-PROTEAN TGX gels are Laemmli-like gels that have a proprietary modification that extends shelf
life to 12 months and enhances separation characteristics relative to conventional gel types. They are
run using standard Laemmli sample buffer and Tris/glycine/SDS running buffer, and they generate
protein migration patterns that are similar to those observed with standard Laemmli Tris-HCl gels.
Two types of TGX formulations are available:
n
Mini-PROTEAN TGX — Laemmli-like, extended shelf life gels
n
Mini-PROTEAN TGX Stain-Free — Laemmli-like, extended shelf life gels with trihalo
compounds that allow rapid fluorescent detection of proteins with the stain-free system,
eliminating staining and destaining steps for faster results (see Chapter 5 for more details)
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Mini-PROTEAN® TGX™ Precast Gels
Instruction Manual and Application Guide
Both gel types gels are available in polyacrylamide single percentages and gradients. Use the protein
migration charts and tables to select the gel type that optimizes resolution of your sample:
n
Use single-percentage gels to separate bands of similar molecular weight. Optimum separation
occurs in the lower half of the gel, so use a percentage in which the protein migrates to the
lower half of the gel
n
Use gradient gels to separate samples containing a broad range of molecular weights. Gradient
gels allow resolution of both high- and low-molecular weight bands on the same gel. Larger
pore sizes at the top of the gel permit resolution of larger molecules, smaller pore sizes toward
the bottom of the gel restrict excessive separation of small molecules
Gel Composition
Crosslinker 2.6% C
Stacking gel 4% T, 2.6% C
Shelf life ~12 months at 2–8°C; expiration date is printed on package
Gel Percentage Optimum Separation Gel Percentage Optimum Separation
Range Range
7.5% 40–200 kD 4–15% 20–250 kD
10% 30–150 kD 4–20% 10–200 kD
12% 20–120 kD Any kD™ 10 –100 kD
Broad Range Unstained
200
116
97.4
21.5
200
116
97.4
66
45
31
21.5
14.4
™
Any kD
4–20%4–15%12%10%7.5%
200
116
66
97.4
45
31
21.5
14.4
6.5
200
116
97.4
66
45
31
21.5
14.4
6.5
200
116
97.4
66
66
45
31
45
31
21.5
14.4
6.5
250
150
100
Precision Plus Protein™ Unstained
™
Any kD
4–20%4–15%12%10%7.5%
250
150
100
75
50
37
250
150
100
75
50
37
25
20
250
150
100
75
50
37
25
20
15
250
150
100
75
50
37
25
20
15
10
75
50
37
25
20
15
10
250
150
100
200
75
50
37
25
20
15
10
116
97.4
66
45
31
Migration charts for protein standards on Mini-PROTEAN TGX and Mini-PROTEAN TGX Stain-Free gels.
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Mini-PROTEAN Precast Gels
3.3 SDS-PAGE Buffers
See Appendix B for buffer formulations. Do not adjust pH.
Running buffer (1x) 25 mM Tris, 192 mM glycine, 0.1% SDS
Dilute 100 ml 10x stock (catalog #161-0732) with 900 ml deionized water (diH2O).
Sample buffer (2x) 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 25% (v/v) glycerol, 0.01% bromophenol
blue, 5% β-mercaptoethanol or 100 mM DTT (added fresh)
Use Laemmli sample buffer (catalog #161-0737) and add β-mercaptoethanol or
DTT before use.
Sample buffer (4x) 250 mM Tris-HCl, pH 6.8, 4% LDS, 40% (w/v) glycerol, 0.02% bromophenol
blue, 15% beta-mercaptoethanol or 200 mM DTT (added fresh)
Use 4x Laemmli sample buffer (catalog #161-0747) and add β-mercaptoethanol
or DTT before use.
3.4 Sample Preparation
1. Determine the appropriate concentration of sample to load (depends on the load volume and the
detection method used; see Chapter 10 for approximate stain sensitivities).
2. Dilute the sample with sample buffer with added reducing agent.
2x: dilute 1 part sample with 1 part sample buffer.
4x: dilute 3 parts sample with 1 part sample buffer.
For nonreducing conditions, omit the reducing agent.
3. Heat the diluted sample at 90–95°C for 5 min or at 70°C for 10 min.
3.5 Running Conditions
Run conditions and times are approximate. Run times represent the time required for the dye front
to reach the line at the bottom of the cassette. Conditions may vary depending on water and buffer
conductivity, which vary from one lab setting to the next. Multiply current by the number of gels run.
Table 3.1. Standard running conditions for SDS-PAGE in the Mini-PROTE AN Tetra cell.
Gel Optimum Range Run Conditions Run Time
7.5% 40–200 kD
10% 30–150 kD 300 V constant:
12% 20–120 kD Starting current (per gel): 55 –75 mA 15–20 min
4–15% 20–250 kD Final current (per gel): 45–70 mA (Fill outer buffer volume
4–20% 10–200 kD to the 4-gel mark)
Any kD 10–100 kD
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