Bio-Rad ReadyPrep Protein Extraction Kit (Soluble/Insoluble) - Manual (Page 12)

10. Centrifuge the tube at maximum speed in a
microcentrifuge (~16,000 x g) for 10–20 min at
18–20°C to pellet any remaining cell debris.
11. Remove and transfer the supernatant (Insoluble
fraction) to a clean tube and discard any residual
pellet.
12. Determine the protein concentration of the Soluble and
Insoluble protein fractions. The Bio-Rad RC DC Protein
Assay (catalog #500-0121 or #500-0122) is
recommended for this measurement. This assay
allows for accurate protein quantitation in the presence
of detergents, reducing agents, and other substances
that typically interfere with other protein assays.
13. An appropriate dilution of each protein fraction will
probably be necessary before IEF/2-D gel analysis.
Refer to Section 6 for guidelines on selecting the
appropriate protein load and volume of sample needed
for a selected IPG strip. For 2-D separation of proteins
from the Insoluble fraction, it is recommended that 2-D
Rehydration/Sample Buffer 1 containing TBP reducing
agent and ampholytes be used for all dilutions. Using
other rehydration/sample buffers that do not contain
ASB-14 detergent may result in some proteins
precipitating out before or during isoelectric focusing.
Additional ReadyPrep 2-D rehydration/sample buffer 1
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