Bio-Rad MicroRotofor™ Cell Lysis Kits - Manual (Page 9)

8. Resuspend 1 g of powdered sample in 3 ml of
prepared PSB. Vortex briefly, then sonicate ten times
for 20 sec each in an ice bath.
9. Centrifuge at 20,000 x g for 30 min at 20°C.
10. Remove any floating debris by filtering the supernatant
using spin filters. If using Bio-Rad's spin filters, centrifuge
at 12–16,000 x g for 10 min. Collect filtrate.
11. 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 in PSB. If
performing the RC DC protein assay, wash the sample
twice. (Optional: A reduction and alkylation of the
sample is recommended at this point. Refer to the
ReadyPrep reduction-alkylation kit, catalog #163-2090).
Note: If applying sample onto an IPG strip (with no
MicroRotofor run to be performed), proceed to step 1,
Section 4 of the ReadyPrep 2-D cleanup kit instruction
manual. If a MicroRotofor step is to be performed, proceed
to step 12. At this point the protein extract can be stored
at –70°C.
Preparing Extracts for a MicroRotofor Run (See
Section 6 of MicroRotofor manual for suggestions of
alternative sample preparation and load conditions.)
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