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

Preparing Extracts for a MicroRotofor Run (See
Section 6 of MicroRotofor manual for alternative sample
preparation and load conditions.)
18. Prepare fresh PSB solution containing PSB diluent,
glycerol, carrier ampholyte, and DTT or TBP (DTT or
TBP is not required if a reduction-alkylation step is
performed at step 17). See Table 2 for recommendations.
19. One MicroRotofor run requires ~2.5 mg protein
(1 µg/µl) in a total volume of 2.5 ml. Using the above
prepared PSB solution, prepare 2.5 ml of a 1 µg/µl
dilution of the protein extract. Load the entire 2.5 ml
sample into the MicroRotofor chamber. It may be
necessary to add extra PSB solution to fill the chamber
completely, eliminating any void volumes.
20. Run the MicroRotofor cell according to the
MicroRotofor instruction manual.
Note: Following fractionaction with the MicroRotofor cell it
is recommended to perform an SDS-PAGE analysis profiling
all 10 fractions. This will illustrate the protein content of
each fraction. See the Appendix for recommendations
pertaining to SDS-PAGE analysis of MicroRotofor fractions.
For subsequent analysis of MicroRotofor fractions by 2-D
PAGE, the ampholyte concentration in samples should not
exceed 0.2–0.5%. If fractions contain high amounts of protein,
dilution prior to loading onto the IPG strip (by 1:10 or greater)
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