Membrane insertion of gap junction connexins: polytopic channel forming membrane proteins

Antipeptide antibodies raised against various regions of cq and ~1 GJ proreins were used for the immunoprecipitation of these proteins from in vitro translation reactions, cell lysates, and subcellular fractions. Antibodies /~IB, ~tE, ~lJ, and BIS are described in Milks et al. (1988) , otlJ and oqS are described in Nishi et al. (1991) , and GAP 10 is described in Rahman and Evans (1991) . Antibody/31 1-6 was raised in rabbits against the first eight amino acids (MNWTGLYT) of~l GJ protein (Risek, 1987) . This antibody cross-reacts with the NH2-terminal domain of cq GJ protein. Specificity of all antibodies was determined by dot-blot analysis as previously described (Milks et al., 1988) . All immunoprecipitations were performed in 1-ml volumes in RIPA buffer consisting of 150 mM NaC1, 0.1% SDS, 1% NP-40, 0.5% deoxycholate, and 50 mM Tris, pH 7.5. 50 ~1 of a 1:10 slurry of protein A-Sepharose (preswollen for 1 h) plus 1-5 txl of antiserum were added to each sample and shaken for 1 h or overnight at 4°C, depending on the strength and specificity of the antibodies. Beads were sedimented by centrifugation and washed t~m times with RIPA buffer before the addition of SDS protein sample buffer.