Bio-Rad Model 225 Tube Gel Casting Stand - Manual (Page 12)

Section 6
Running the First-Dimension (IEF)
6.1 Electrode Preparation
1. Insert a gel tube with reservoir or a stopper into each of the 16 positions in the
tube adaptor. To insure that the upper buffer does not leak into the lower buffer
chamber, the sample reservoirs and stoppers must be firmly inserted into the
tapered holes in the tube adaptor. Insert the stoppers with a twisting motion.
2. Fill each sample reservoir with thoroughly degassed upper buffer chamber
electrolyte. Using a sample loading syringe with needle, e.g., 20 µl Hamilton
syringe, expel any air bubbles from the capillary space in the neck of the
sample reservoir and in the flexible tubing connector.
3. Place a 1 inch stir bar in the lower chamber, and place the tube gel adaptor into
the lower buffer tank. Fill the lower tank with electrolyte. The maximum buffer
level in the lower buffer chamber should be enough to bring it about even with
the blue line on the glass gel tubes. This corresponds to an approximate volume
of 800 ml. If the buffer level is higher than the blue lines on the gel tubes, an
electrical short circuit may be created, which will prevent electrofocusing and
may damage the equipment. Check the bottom of each tube for trapped air
bubbles. Bubbles can be removed using a Pasteur pipet with a curved tip.
4. Fill the upper buffer chamber of the tube adaptor module. The buffer level in
the upper buffer chamber should be slightly above the sample reservoirs but
below the top of the plastic bar under which the platinum electrode is mounted.
This is a volume of approximately 60 ml. If the upper buffer level is too high,
there may be an electrical short circuit created between the banana plug
connector for the lower chamber and the liquid in the upper chamber. Before
operating the unit, make sure there is no moisture around the banana plug
connectors which might create a short circuit. Dry these areas with a paper
towel if necessary.
Note: It is important not to overfill the buffer chambers in the 2-D cell. If the
chambers are overfilled, an electrical short circuit can occur with the upper
chamber causing excessive current to be drawn from the power supply. This
may damage the tube adaptor due to overheating.
5. Place the lid on the cell. Match the red lead on the lid with the red coded post
on the tube adaptor.
6. Place the unit of a magnetic stirring motor, and stir slowly. This will provide
cooling for the heating, which can occur in the capillary tubes.
7. Connect the cell to the power supply and pre-electrophorese the tube gels by
running at 200 V for 10 minutes, 300 V for 15 minutes, and 400 V for 15 minutes.
The Bio-Rad PowerPac 3000 and PowerPac 1000 power supplies are
recommended for this application. These steps are performed automatically
when the unit has been programmed for this operation.
Note: Pre-electrophoresis, as described in steps 5, 6, and 7 of Section 6.1, is
optional. The original O'Farrell protocol uses pre-electrophoresis. Other
protocols indicate it is not required.
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