Methods to separate nuclear soluble fractions reflecting localizations in living cells

To understand various intranuclear functions, it is important to know when, what, and how proteins enter the nucleus. Although many methods and commercial kits for nuclear fractionation have been developed, there are still no methods for obtaining a complete nuclear proteome. Soluble nuclear proteins are often lost during fractionation. We developed remarkably improved methods to obtain nuclear soluble fractions by optimizing the conditions of selective permeabilization of the plasma membrane. As a result, 10 million cells could be separated into the cytoplasmic and nuclear soluble fractions more precisely in a 1.5-mL test tube. Moreover, the addition of an inhibitor to prevent leakage from the nucleus retained small proteins in the nucleus. Because of the simple protocols and easy application for multiple samples, our methods are expected to be applied to various studies on spatiotemporal changes of dynamic nuclear proteins, such as signal transduction.