Bio-Rad ReadyPrep Protein Extraction Kit (Cytoplasmic/Nuclear) - Manual (Page 10)

8. Suspend the nuclei in 0.5 ml of complete PSB. Vortex
the tube 4–5 times, 60 sec each, to solubilize the
nuclear proteins. Genomic DNA should be evident in
the sample tube at this stage. Centrifuge at maximum
speed (12–16,000 x g) for 15–20 min at room
temperature to pellet the genomic DNA and other
debris and transfer the clarified supernatant into a new
microcentrifuge tube labeled Nuclear Protein Fraction.
9. Reextract the residual pellet from step 8 using
approximately one-fourth to one-half the volume of
complete PSB used previously (0.13–0.25 ml).
Centrifuge at maximum speed for 15–20 min at room
temperature, collect the clarified supernatant, and pool
it with the previous nuclear protein fraction.
4.1.2 Processing Cytoplasmic and Nuclear Protein
Fractions for IEF/2-D Analysis
10. Determine the protein concentration of the the
cytoplasmic and nuclear protein fractions. The RC DC
protein assay is recommended for this measurement.
This assay allows accurate protein quantitation in the
presence of detergents, reducing agents, and other
substances that typically interfere with other protein
assays.
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