Multimer analysis of rVWFR273W secreted by COS-7 cells.

The cells were transfected with the appropriate plasmid DNA then placed in serum-free medium 24 hours after transfection. Medium supernatants were harvested after an additional 48 hours. An appropriate volume of medium containing 1 mU of rVWF wild-type as determined by ELISA was analyzed on a nonreducing 2% SDS-agarose gel (lane 1). Ten milliunits of each rVWF as determined by ELISA was immunoprecipitated with rabbit antihuman VWF polyclonal antibody, then analyzed on a nonreducing 2% SDS-agarose gel (lane 2 wild-type rVWF, lane 3 rVWFR273W). Multimers were visualized using polyclonal rabbit antihuman VWF antibody and a secondary HRP-conjugated swine antirabbit IgG polyclonal antibody and enhanced chemiluminescence.