Bio-Rad MicroRotofor™ Cell - Manual (Page 10)

Note: Keep the vacuum valve closed until the run is completed and you are
ready for harvesting the fractions.
8. Turn the power switch to the “ON” position to start the oscillating motor.
9. Set the cooling switch to setting II (20°C).
(Note: See Table 3.2 in the MicroRotofor instruction manual, for detailed
cooling setting information.)
10. Attach the leads on the lid to a PowerPac HV power supply or other
commercial power supply capable of power control at 1 W. If your power
supply cannot be set to run under constant power, run the instrument in
a stepwise constant voltage mode with limiting current (see Section 4.5
for detailed power conditions.)
11. A typical run is completed in less than 3 hours. To monitor the focusing
progress, observe the voltage increase over time. The run is complete
when the voltage stabilizes. At that point, allow the run to continue for
30 minutes before harvesting. Longer run times do not improve focusing
and may result in a collapse of the pH gradient.
12. The progress of the run can also be monitored by following the migration
of the colored proteins in the sample.
Fig. 10. Migration of colored proteins.
• Phycocyanin has three blue subunits of pI 4.5, 4.7 and 5.0 that
migrate towards the anode and are expected to focus in fraction # 3.
• Hemoglobin A and Hemoglobin C, two red colored proteins of pI 7.1
and 7.4, respectively, migrate towards the center of the focusing
chamber and are expected to focus in fraction # 6 and/or #7.
• Cytochrome c, an orange protein of pI 9.6 migrates toward the
cathode and is expected to focus in fraction # 9 and/or 10.
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