Figure 5
From: Crude subcellular fractionation of cultured mammalian cell lines
Application of the approach to other cell lines. (A) HT1080 cells (2 × 105) and HeLa cells (1 × 105) were plated in the wells of a 12 well plate and the digitonin concentration required for optimal extraction of the cytosol was determined as in Figure1. (B) The remainder of the protocol from Figure 2 was then applied using 100 μg/ml digitonin as the optimal cytosolic extract in each case. An aliquot of each extract was then analyzed by 4-12% SDS PAGE followed by Western blotting and probing with antibodies to GAPDH, BiP and Histone H3 (Used as an alternative nuclear marker due to weakness of the Lamin A signal in HeLa cells). (C) Samples were analyzed by 4-12% SDS PAGE followed by staining with Coomassie blue and then silver staining showing the difference in protein composition of each sample. The bracket alongside the gels marks the position of Histones in the RIPA nuclear extract and the asterisk next to the silver stained gels denotes protein bands unique to the E-RIPA extracts.