The analysis of Bcl-2 family proteins by immunoblotting involves: (a) lysis of cells or processing of tissues for extraction and recovery of intact proteins; (b) SDS-PAGE separation of proteins and transfer to either nitrocellulose or nylon membranes; and (c) detection of antigens using antibody-based methods. In addition, the reprobing of blots with additional antibodies is often an important consideration. Here, we describe methods which have provided successful for extraction of Bcl-2 family proteins from cultured cells and tissues, and for immunodetection of these proteins once immobilized on membranes. A procedure developed in our laboratory which permits multiple reprobings of blots is also described (10).
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