Bio-Rad ReadyPrep™ Protein Extraction Kit (Membrane II) - Manual (Page 14)

14. 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.
15. Remove and transfer the supernatant (Membrane
Protein fraction) to a clean tube and discard any
residual pellet.
16. Determine the protein concentration of the membrane
protein sample. 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.
17. An appropriate dilution of the membrane protein
fraction may 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 the
membrane proteins, 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 precipi-
tating out before or during isoelectric focusing.
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