Using Dounce Homogenization to Lyse Cells for Immunoprecipitation.

Dounce homogenization in combination with hypotonic buffers facilitates the lysis of adherent and suspension cells. Addition of hypotonic buffers results in the swelling of the cell's cytoplasm, allowing for the gentle rupture of cell membranes by mechanical force. Dounce homogenization releases cytoplasmic proteins that can be processed separately from the remaining intact nuclei, which can undergo high-salt extraction for detergent-free extraction of nuclear proteins. In this protocol, cells are initially swollen by incubation in hypotonic buffer, making them susceptible to dounce lysis. Douncing is continued until most cells are lysed, leaving free intact nuclei behind from which nuclear proteins can be extracted. This method does not facilitate the extraction of histones; however, it is effective in extracting transcription factors and other chromatin-bound proteins. It is important to keep buffer volumes to a minimum to maintain high protein concentrations. Upon completion of hypotonic and high-salt extraction, respective cytoplasmic and nuclear fractions undergo dialysis to achieve physiological salt conditions before further use.