Bio-Rad ReadyPrep Protein Extraction Kit (Cytoplasmic/Nuclear) User Manual
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3. For each 0.05 ml of packed cells, add 0.5 ml of ice-
cold CPEB. Vortex to suspend the cell pellet and
incubate the cells on ice for 30 min.
Notes: Protease inhibitors may be added to CPEB
immediately prior to use to prevent proteolysis during the
extraction process.
Insufficient volume of CPEB may result in poor cell lysis,
low cytoplasmic protein yield, and contamination of the
nuclear pellet with cytoplasmic proteins.
4. Gently pass the cell suspension through a narrow-
opening syringe needle (20 gauge) to lyse the cells
without damaging the nuclei. Slowly force the cells
through the needle for 10–20 strokes to ensure
complete cell lysis.
Note: Care should be exercised when breaking open the
cells; excessive handling and severe force may damage the
nuclei, releasing nuclear proteins into the cytoplasmic
fraction. Some optimization of the handling procedure may
be required.
5. Centrifuge the cell lysate at 1000 x g for 10 min at
4°C. Upon completion of the centrifugation, use a
pipet to immediately transfer the supernatant
containing the cytoplasmic proteins to a new tube
(on ice) labeled Cytoplasmic Protein Fraction.
Centrifuge the original tube for 5–10 sec at 1000 x g,
remove any remaining liquid, and pool it with the
previous supernatant.
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