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.

Assembly

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.

Preparing Samples

1. Add 50 µL of bME to 950 µL 3X loading dye.
2. Heat to 95° C for 5 minutes.
3. Load samples into wells once they have cooled to RT.

Loading The Samples

1. Prepare 300 ml of electrode buffer by combining 30 ml of 10X electrode buffer with 270 ml of deionized water.
2. 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 the electrophoresis.
3. 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.
4. 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

1. 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.
2. Attach the electrical leads to a suitable power supply (200 V minimum) with the proper polarity.
3. 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

1. After electrophoresis is complete, turn off the power supply and disconnect the electrical leads.
2. Remove the cell lid and carefully pull the inner cooling core out of the lower chamber. Pour off the upper buffer.
3. Lay the inner cooling core on its side and remove the gels and the attached clamp assembly.
4. Loosen all four screws of the clamp assembly and remove the gel.
5. Push one of the spacers of the gel out to the outside of the plates without removing it.
6. 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.
7. 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

Lab Schedule In Context of Research Project