Owner manual

15. One MicroRotofor 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
rat brain 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.
16. Run the MicroRotofor cell according to MicroRotofor
manual instructions — typically 1500 Vhs at 1 Watt
(constant) when using PSB solution as recommended
in this kit.
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. 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) is recommended.
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