Bio-Rad ReadyPrep Protein Extraction Kit (Cytoplasmic/Nuclear) - Manual (Page 9)

6. The pellet in the tube from step 5 contains nuclei. Wash
the nuclear pellet one time with 0.25 ml of CPEB (see note
below). Vortex briefly or gently pipet up and down to
resuspend the nuclei and incubate on ice for 10 min.
Centrifuge as in step 5 to concentrate the nuclei and
either pool the wash supernatant with the previous
Cytoplasmic Protein Fraction or discard.
Notes: Washing of the nuclei is critical to minimize
contamination of the nuclei with cytoplasmic proteins. If
desired, step 4 can be repeated after resuspending the
nuclear pellet to ensure that all cells have been broken
open and that cytoplasmic proteins are not introduced into
the nuclear fraction via cellular contamination of the
nuclear pellet. Insufficient washing of the nuclei can lead to
failure in the enrichment of nuclear proteins and result in
2-D gels where the cytoplasmic and nuclear fractions
produce very similar protein spot patterns.
If no further downstream applications are to be immediately
performed, aliquot and quick-freeze the cytoplasmic protein
fraction on dryice and then store at -70°C. Also, to avoid
repeated freeze-thawing of the sample, it is often
convenient at this stage to set aside a 5–10 µl aliquot of the
cytoplasmic protein fraction before freezing for protein
quantitation. This amount is sufficient for the Bio-Rad RC
DC protein assay.
7. Prepare a fresh volume of complete PSB sufficient to
suspend the nuclear pellet formed in step 6 (a total of
0.75 ml is needed per 0.05 ml of original packed cells).
See Section 5.2 for instructions on preparing the
complete PSB.
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