How to Perform a Pre-Cast SDS-PAGE
Note: The upper stacking gel and sample buffer
have a pH of 6.8 while the lower resolving gel has a pH of 8.8. The running
buffer (a solution of Tris and glycine) is put in both buffer chambers
and has a pH of 8.3.
Assembling the Upper Buffer Chamber
These are the directions that come with the apparatus
and they read like directions of how to tie a shoe - not well. Nevertheless,
we need some written directions to insure a leakproof seal, make sure the
gray U-shaped inner cooling core gaskets are clean. Inspect the gasket for
small cuts that could result in an upper buffer leak. There are two sides
to this gasket. Make sure that the side with the notch is exposed for contact
with the gel sandwich.
Lay the inner cooling core flat on a lab bench.
With the glass plates of the gel sandwich facing the cooling core (and the
clamp screws facing out), carefully slide the clamp assembly wedges underneath
the locator slots on the inner cooling core until the inner glass plate
of the gel sandwich butts up against the notch in the U-shaped gasket.
Lubricating the raised portions of the U-shaped gasket with a drop of running
buffer or water helps the glass plate sandwich slide properly.
While pushing the clamp assembly slightly up toward
the top of the locator slots, snap the clamp assembly fully onto the cooling
core by pressing at the bottom of the clamp assembly until the cooling core
latch engages each side of the clamp assembly. (Do not pull out on cooling
core latch at the same time.) It is very important that there is good contact
at the point where the short glass plate contacts the notch in the gasket
to prevent upper buffer leaks. If the glass plate is not firmly seated in
its proper place against the notch in the gasket buffer leaks will occur.
Turn over the inner cooling core and attach another
clamp assembly to the other side of the core in the same manner.
- Add 50 µL of bME
to 950 µL 3X loading dye.
- Heat to 95° C for 5 minutes.
- Load samples into wells once they have cooled to RT.
Loading The Samples
- Prepare 300 ml of electrode buffer by combining
30 ml of 10X electrode buffer with 270 ml of deionized water.
- Lower the inner cooling core into the lower buffer
chamber of the Mini-PROTEAN II cell. Add approximately 115 ml of buffer
to the upper buffer chamber. Fill until the buffer reaches a level halfway
between the short and long plates. Do not overfill the upper buffer chamber.
Overfilling the upper buffer chamber will result in siphoning of the buffer
over the top of the gasket, resulting in buffer loss and interruption of
- Pour the remainder of the buffer into the lower
buffer chamber so that at least the bottom 1 cm of the gel is covered.
Remove any air bubbles from the bottom of the gel so that good electrical
contact is achieved. This can be done by squirting the lower buffer with
a curved pipette until the bubbles clear.
- Load the samples into the wells under the electrode
buffer with a pipette and one very thin tip. Insert the tip to about 1-2
mm from the well bottom before delivery.
Running The Gel
- Place the lid on top of the lower buffer chamber
to fully enclose the cell. the correct orientation is made by matching
the colors of plugs on the lid with the jacks on the inner cooling core.
- Attach the electrical leads to a suitable power
supply (200 V minimum) with the proper polarity.
- Apply power to the Mini-PROTEAN II cell and begin
electrophoresis. The recommended power for optimal resolution with minimal
thermal band distortion is 200 volts, constant voltage setting. No adjustment
of the setting is necessary for spacer thickness, or number of gels. The
usual run time is approximately 45 minutes. Current should be approximately
60 mA per gel (120 mA for two gels) at the beginning of the run. During
the 45 minute run the current will slowly drop to about 30 mA per gel.
This drop is caused by the change in buffer ions in the gel, causing a
slow rise in the resistance in the gel.
Removing The Gel
- After electrophoresis is complete, turn off the
power supply and disconnect the electrical leads.
- Remove the cell lid and carefully pull the inner
cooling core out of the lower chamber. Pour off the upper buffer.
- Lay the inner cooling core on its side and remove
the gels and the attached clamp assembly.
- Loosen all four screws of the clamp assembly
and remove the gel.
- Push one of the spacers of the gel out to the
outside of the plates without removing it.
- Gently twist the spacer so that the upper glass
plate pulls away from the gel. Remove the plate. The gel will stick to
one of the plates.
- Float the gel off of the glass plate by inverting
the gel and glass plate under fixative solution or blotting transfer buffer
and agitate gently until the gel separates from the glass plate.
4X sample buffer - store at RT°
- 6.06 g Tris
- 2 ml 20% SDS
- 20 ml glycerol
- pH to 6.8 with HCl
- water to 100 ml
- 0.03% bromophenol blue
- plus fresh DTT added just before use
Lab Schedule Outlined
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