An optimized protocol based on the instructions of a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology, P0027) was used to separate the nuclear portion from the cytoplasmic portion. Cells were digested with a 0.02% EDTA solution at 37 °C for 2 min and centrifuged at 4 °C for 5 min. The precipitate was lysed by cytosolic protein extraction reagent A containing 1% PMSF for 10–15 min on ice. Cytosolic protein extraction reagent B was then added, and the suspension was kept on ice for another 5 min. After centrifugation, the supernatant (containing cytoplasmic proteins) was transferred to a precooled tube. The pellet was washed once with a mixed solution of cytosolic protein extraction reagents A and B (20:1), and SDS-PAGE sample loading buffer containing SDS was added to lyse the nuclei.
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