Differential Detergent Fractionation of Eukaryotic Cells

Abstract

INTRODUCTIONDifferential detergent fractionation (DDF) involves the sequential extraction of cells with PIPES buffers containing first digitonin, then Triton, and finally Tween/deoxycholate (DOC). The procedure yields four biochemically and electrophoretically distinct fractions composed of the following: (1) cytosolic proteins and extractable cytoskeletal elements; (2) membrane and organelle proteins; (3) nuclear membrane proteins and extractable nuclear proteins; (4) detergent-resistant cytoskeletal filaments and nuclear matrix proteins. Most of these fractions can be enriched or purified further by subsequent manipulations. The DDF protocol described here represents a modification of a method used for the fractionation of Madin-Darby canine kidney cells, including the addition of a digitonin extraction step, the inclusion of EDTA in the digitonin and Triton buffers, and the elimination of a nuclease digestion step; DNA is instead denatured by shear force in the presence of sodium dodecyl sulfate (SDS).