Mini SDS-PAGE Gel Protocol for the Bio-Rad Mini-Protean Tetra Cell
Note: For best results, use MilliQ or nanopure equivalent water to prepare all buffers and
samples.
Preparing 1X SDS PAGE Running Buffer
10X Tris Glycine SDS PAGE Running Buffer
Components
Tris
Glycine
SDS
Nanopure water
Amount for 1 L
30.3 g
144.1 g
10 g
Adjust to 1 L
Dilute to 1X for use. The 1X can be made several days in advance. You will need 800 ml buffer for 1-2 gels
and 1200 ml for 3-4 gels.
Note: If the 10X or 1X buffer is turning yellow, the glycine is degrading and so it is time to properly
dispose of it.
Sample Preparation
1) Prepare the samples:
10-Well Gels- maximum loading volume 30 µl/well (1.0 mm thick)
15-Well Gels- maximum loading volume 15 µl/well (1.0 mm thick)
Sample
Water
Laemmli Sample Buffer (2X)
Β-mercaptoethanol
Total Volume
10 μl
2.0 μl
10 μl
2) Prepare 1X sample buffer to fill the empty wells:
Water
Laemmli Sample Buffer (2X)
Β-mercaptoethanol
Total Volume
7.5 μl
1.5 μl
15 μl
10 μl
2.0 μl
20 μl
12.5 μl
2.5 μl
25 μl
15 μl
3 μl
30μl
400 μl
500 μl
100 μl
1000 μl
3) Incubate sample(s) at 95°C for 5 min.
Note for people who are working with membrane proteins: Do not boil your sample because it
may cause your proteins to aggregate. Instead, load your sample immediately after adding the
loading buffer.
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Preparing a Precast Gel for Electrophoresis
1)
2)
3)
4)
Remove the precast gel from the storage pouch.
Rinse the outside of the gel thoroughly with distilled water.
In one fluid motion, gently remove the comb.
Rinse the wells thoroughly with either: distilled water or 1X running buffer. Invert the gel and shake to
remove excess buffer/water. Repeat twice. If necessary, straighten the sides of the wells.
5) Remove the tape from the bottom of the Precast Gel cassette to expose the bottom edge of the gel.
6) Repeat for any remaining gels.
Assembly of the Mini-Protean Tetra Cell
1) On a clean flat surface, open the clamping frame on the Electrode Assembly (see Figure 4a below).
When running only 1-2 gels, use the Electrode Assembly (the one with the banana plugs), NOT the
Companion Running Module (the one without the banana plugs).
2) Place the first gel cassette (with the short plate facing inward) onto the gel supports molded into the
bottom of the clamping frame assembly surface (see Figure 4a). Note that the gel will now rest at a
30° angle, tilting away from the center of the clamping frame (see Figure 4b).
Note: Please use caution when placing the first gel. Make sure that the clamping frame remains
balanced and does not tip over.
3) Place the second gel on the other side of the clamping frame, again by resting the gel onto the
supports (see Figure 4b). If running an odd number of gels (1 or 3), use the buffer dam. At this point
there will be two gels resting at an angle, one on either side of the clamping frame, tilting away from
the center of the frame.
4) Using one hand, gently pull both gels towards each other, making sure that they rest firmly and
squarely against the green gaskets that are built into the clamping frame.
5) While gently squeezing the gel cassettes 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.
Alternatively, you may choose to pick-up the entire assembly with both hands. While making sure that
the gels do not shift, simultaneously slide both arms of the clamping frame into place (see Figure
4c).
6) Check to make certain that the short plates sit just below the notch at the top of the green gasket.
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7) Place the Electrode Assembly on the bench top. Fill the inner chamber on the Electrode Assembly
with 1X SDS PAGE Running Buffer (Figure 4d). The buffer should be just under the top edge of the
outer gel plate (200 ml). Check to see that the buffer is not leaking from the inner chamber onto the
bench top.
Gel Supports
Put buffer here
Sample Loading
1) Using a gel loading pipette tip, load the molecular weight standards (usually in lane 1).
2) Load your samples into the appropriate wells by lowering the pipette tip to the bottom of the sample
well and slowly pipet the sample into well. Be careful not to contaminate another well with the sample
or puncture the bottom of the well.
3) Load 20 µl 1X Sample loading buffer into any remaining empty wells. This promotes uniform running of
the stacking front.
Running Gel(s)
1) Place the tank with the front of the tank facing you (the front of the tank is the face that has the 2-Gels
and 4-Gels line markings). When oriented properly, the red marking on the top inside edge of the tank
will be on your right, and the black marking on the top inside edge of the tank will be on your left.
2) Place the Electrode Assembly (banana plugs) so that this assembly is in the back position of the cell,
making sure that the red (+) electrode jack matches the red marking on the top right inside edge of the
tank.
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3) If running 3-4 gels, place the the Companion Running Module (no banana plugs) in the front position.
Make sure again that red (+) electrode is matching with the red marking on the top inside right edge of
the tank.
CAUTION: When running only 1-2 gels, DO NOT place the Companion Running Module in the
tank. Doing so will cause excessive heat generation and prevent electrophoretic separation.
4) Fill the tank (lower chamber) with 1X SDS PAGE Running Buffer to the indicated level (550 ml for 2
gels and 680 ml for 4 gels).
5) Place the Lid on the Mini-PROTEAN Tetra Tank. Make sure to align the color coded banana plugs and
jacks. The correct orientation is made by matching the jacks on the lid with the banana plugs on the
electrode assembly.
6) Insert the electrical leads into a suitable power supply with the proper polarity.
For 1-2 gel: PowerPac Basic or PowerPac 200
For 3-4 gels: use any PowerPac Basic or PowerPac HC
7) Apply power to the Mini-PROTEAN Tetra Cell and begin electrophoresis:
Standard Method
200 V until the dye front reaches the bottom (expected run time 31-39 minutes)
Low Voltage Method-Membrane Proteins and Bands for Mass Spectrometry Analysis
40 V for 10 min (allows better stacking of the proteins)
100 V until the dye front reaches the bottom (expected run time 80-90 minutes)
Note: The low voltage method reduces the amount of heat which is generated during
electrophoresis resulting in sharper bands. For further cooling, the gel maybe ran in a
coldroom/coldbox. Don’t prechill the running buffer since colder temperatures will cause
the SDS to come out of solution.
Gel Removal
1) At the completion of the run, turn off the power supply and disconnect the electrical leads.
2) Remove the tank lid and carefully lift out the electrode assemblies. Pour off and discard the running
buffer into the proper hazardous waste container.
Note: Always pour off the buffer before opening the arms of the assembly to avoid spilling the
buffer.
3) Open the arms of the assembly and remove the gel cassettes.
4) Open the cassette by aligning 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.
5) Hold the cassette plate over a container with water or transfer buffer with the gel side facing towards
the container. Place the gel into the fluid and agitate gently until the gel separates from the plate. You
may need to use Gel Knife to carefully loosen the lower corner of the gel and allow the gel to peel away
from the plate.
6) Rinse the Mini-PROTEAN Tetra cell electrode assembly, clamping frame, and mini tank with distilled,
deionized water.
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References
The protocol above was based in part, on the following literature from Bio-Rad:
• Mini-PROTEAN Precast Gels: Instruction Manual and Application Guide (1658100 Rev A)
• Mini-PROTEAN TGX and TGX Stain-Free Precast Gels: Quick Start Guide (Bulletin 5933 US/EG
RevB)
• Mini-PROTEAN TGX Precast Gels (Bulletin 5871)
Supplies
Below lists the supplies needed. Comparable supplies may be purchased through other vendors; however, the
gels need to specifically fit the Bio-Rad Mini-Protean Tetra Cell.
Gels
Mini-Protean TGX Precast Gels, Any kD, 10 well, 30 µl
Mini-Protean TGX Precast Gels, Any kD, 10 well, 30 µl
Mini-Protean TGX Precast Gels, Any kD, 10 well, 50 µl
Samples Buffer Reagents
Laemmli Sample Buffer
2-Mercaptoethanol
Protein Standards
Precision Plus Protein Unstained Standards (Sypro Ruby)
Precision Plus Protein Dual Color Standards (Western)
BenchMark Unstained Protein Ladder (Sypro Ruby)
BenchMark Prestained Protein Ladder (Western)
Stains
Sypro Ruby Fluorescent Protein Stain (10 ng sensitivityovernight)
Gelcode Blue Stain Reagent (25 ng sensitivity-overnight)
Size
Vendor
Catalog
Number
2 gels/box
10 gels/box
10 gels/box
Bio-Rad
Bio-Rad
Bio-Rad
456-9033S
456-9033
456-9034
30 ml
25 ml
Bio-Rad
Bio-Rad
161-0737
161-0710
1 ml
500 µl
2 x 250 µl
2 x 250 µl
Bio-Rad
Bio-Rad
Life Technologies
Life Technologies
161-0363
161-0374
10747012
10748010
200 ml
Life Technologies
S12001
500 ml
Thermo Scientific
Pierce
24590
Homemade Coomassie Blue (100 ng sensitivity)
Imperial Protein Stain (>10 ng sensitivity)
200 ml
Thermo Scientific
Pierce
Bio-Rad
161-0495
2.5 L
4L
EMD/VWR
EMD/VWR
EMD-AX0073-9
EM-MX0475-1
1L
Oriole Fluorescent Gel Stain
Fixer/Destain Solution
Acetic Acid
Methanol
Written by Melissa Sondej (version 04/17/12)
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