Bio-Rad Model 225 Tube Gel Casting Stand User Manual
Page 13
6.2 Sample Preparation
Add an equal volume of first-dimension sample buffer to the sample. Incubate at
room temperature for 10–15 minutes. The sample reservoirs can accommodate
volumes up to 100 µl, but smaller volumes are recommended, e.g., 25 µl. An
optimum protein load for a complex sample is about 5–10 µg protein, though high-
er protein loads can be applied with satisfactory results. For purified proteins, the
recommended amount is about 1–5 µg. If the spots are detected with silver stain,
or SYPRO Ruby protein gel stain smaller amounts of sample may be satisfactory.
6.3 First-Dimension (IEF) Sample Loading
1. After pre-electrophoresis, discard the upper and lower chamber buffers.
Remove the pre-electrophoresis solutions from the sample reservoirs and the
tube gels with the Hamilton syringe. Fill the gel tubes and the sample reservoirs
with freshly degassed upper chamber buffer. Remove all bubbles with the
Hamilton syringe.
2. To load sample, use a sample syringe with a blunt needle. Add sample at the
top surface of the gel.
3. Overlay the sample with 20-40 µl sample overlay buffer. This buffer must have
an intermediate density between the sample and the electrolyte. It may be
necessary to dilute the sample overlay buffer with a small volume of distilled
water.
4. Set up the apparatus as before (Section 6.1 steps 4–8). Do not disturb the
sample while filling the upper buffer chamber with electrolyte solution.
6.4 Power Conditions
1. Run at 500 V for 10 minutes.
2. Increase the voltage to 750 V for 3.5 hours. Higher voltages are not recommended
because the unit may overheat.
Note: If the first-dimension (IEF) run is done at lower voltages, the run time must
be increased. For example, when IEF is done at 400 V the run will be completed
in approximately 6 hours. The times given here are approximate and the IEF run
time must be determined for each sample. To determine the optimum IEF run time
at a given voltage, set up eight tube gels with the same sample. Remove two
tubes every 2 hours, and run the tube gels in the second dimension according to
the directions below. A comparison of the results should provide the information
needed to determine the optimum IEF run time.
10