Bio-Rad ReadyPrep Protein Extraction Kit (Signal) - Manual (Page 10)

7. The final pellet contains the signal proteins. Resuspend
the pellet in 0.2–0.3 ml of complete PSB. Vortex the
tube 4 to 5 times for 60 seconds each. Incubate the
tube at room temperature for 10–15 min vortexing
periodically to ensure the pellet is completely
dissolved. Centrifuge the sample at maximum speed in
a microcentrifuge for 10 minutes at room temperature
and collect the clarified supernatant. To improve
recovery of the signal proteins, reextract any residual
pellet with about one-third the original volume of
complete PSB and combine the supernatant with that
obtained previously. See Section 5.2 for instructions
on preparing the complete PSB.
8. Determine the protein concentration of the signal
proteins. The Bio-Rad RC DC Protein Assay 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. Make
an appropriate dilution of the resuspended signal
proteins into complete PSB or other strongly
chaotropic 2-D rehydration/sample buffer before
IEF/2-D gel analysis. Refer to Section 5.1 for
guidelines on selecting the appropriate volume of
buffer to use.
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