Bio-Rad MicroRotofor™ Cell Lysis Kits User Manual
Page 11
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) will be sufficient to reduce the ampholyte
concentration. In cases where protein levels are lower, use
of the ReadyPrep 2-D cleanup kit (catalog #163-2130) for
ampholyte removal is recommended.
Section 5
Appendix
Preparation for SDS-PAGE
CHAPS, a component of the PSB diluent, may interfere
with SDS-PAGE. Remove CHAPS from the extracts (for
example, with the ReadyPrep 2-D cleanup kit) or dilute the
extracts 1:1 with 1x Laemmli buffer prior to SDS-PAGE.
Preparation for IEF on an IPG Strip
The sample extract can be loaded directly onto an IPG
strip after appropriate dilution. See Table 3 for
recommendations on how much sample to load onto an
IPG strip. Dilution of the sample can be done using the
protein solubilization buffer (PSB) as a rehydration/sample
buffer. However, some critical components need to be
added to the PSB solution to make it IEF-compatible (Table 2).
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