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

12. 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 11). See Table 2 for recommendations.
13. One MicroRotofor run requires ~2.5 mg protein
(1 µg/µl) in a total volume of 2.5 ml. Using the above
prepared solution, prepare 2.5 ml of a 1 µg/µl dilution
of the protein extract. These recommendations are
based on spinach leaf as a sample source and may
vary depending on sample type. 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.
14. Run the MicroRotofor cell according to the
MicroRotofor instruction manual (typically 1500 Vh at
1 Watt constant). After the run, harvest the fractions
and proceed to step 1, Section 4 of the ReadyPrep
2-D cleanup kit instruction manual. This is important
for removal of phenolics and high concentrations of
carrier ampholytes.
Note: Following fractionation 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.
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