Owner manual

11. Transfer the supernatant carefully into a new tube
without disturbing the pellet.
12. Resuspend the residual cell pellet in 250 µl of prepared
PSB, and repeat steps 9 and 10. Collect the
supernatant and pool with the first supernatant.
13. Determine the protein concentration of the extract.
This is best done using the RC DC protein assay
(catalog #500-0121 or 500-0122), which is compatible
with the detergents and reducing agents present in
PSB. If performing the RC DC protein assay, keep in
mind that two washes of the sample are recommended.
(Optional: A reduction and alkylation of the sample is
recommended at this point in the procedure. Refer to
the ReadyPrep reduction-alkylation kit, catalog
#163-2090). Store the protein extract at –70°C, apply
it directly onto an IPG strip (see Appendix for details),
or proceed to step 14.
Preparing Extracts for a MicroRotofor Run (See
Section 6 of the MicroRotofor manual for suggestions of
alternative sample preparation and load conditions.)
14. 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 13). One MicroRotofor run requires
2.5 ml sample. See Table 2 for recommendations.
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