Subcellular Fractionation: Cytoplasmic and Nuclear Isolation

All preparations were performed on ice. Cells were resuspended in 1 mL hypotonic solution containing 0.1% NP-40 and incubated for 3 min. Next, cells were homogenized using a Potter-Elvehjem homogenizer by ~20 iterations of up and down passes of the pestle. The solution was centrifuged to pellet nuclei (1000 rcf, 5 min). Supernatant (cytoplasmic fraction) was re-centrifuged (15,000 rcf, 3 min) to pellet debris. Fractionation with non-ionic detergents was carried out by adding a hypotonic solution to the cells for 3 min. Then, NP-40 or digitonin were added to a final concentration 0.1%. The resulting solutions were kept for 3 min and centrifuged (1000 rcf, 5 min). Supernatant (cytoplasmic fraction) was re-centrifuged (15,000 rcf, 3 min) to sediment debris.
Fractionation by the L&W method and its variations (including those with DNase I addition and subfractionation using RIPA-buffer) is summarized as a step-by-step protocol in the section “The L&W nucleus/cytoplasm fractionation protocol.” The compositions of the hypotonic, isotonic, DNase I, and RIPA buffers are also given in the same section.