Interaction of murine fac with GRP94 in vivo. Liver cytoplasmic extracts from C57B/6 mice were subjected to immunoprecipitation with either polyclonal anti-FAC antibody or monoclonal anti-GRP monoclonal antibody. The anti-FAC antibody can bind efficiently protein A, whereas the rat anti-GRP94 antibody binds well to protein G, but not to protein A. Thus, to increase the recovery of immune complexes and minimize quantitative differences that may result from such interactions, immune complexes were precipitated with a mixture of protein A-agarose and protein G-agarose. After electrotransfer to PVDF membranes, the expression of GRP94 was detected using anti-GRP94 antibody (1 μg/mL), HRP-conjugated goat antirat IgG, and EC. Lane 1, 50 μg extract analyzed directly without prior immunoprecipitation; lane 2, 250 μg extract incubated with protein A-agarose and protein G-agarose without primary antibodies; lane 3, 250 μg extract immunoprecipitated with anti-FAC antibody (4 μg in 250 μL extract) and subsequently with protein A-agarose and protein G-agarose; 250 μg extract immunoprecipitated with anti-GRP94 (4 μg in 250 μL extract) antibody and subsequently with protein A-agarose and protein G-agarose. The positions of murine GRP94 and rabbit IgG heavy chain are shown (arrow).