Coimmunoprecipitation of band 3 and Rh proteins from human RBC membranes.

RBC membrane proteins were solubilized in a deoxycholate-containing buffer and were incubated with protein G-Sepharose, which had been preloaded with mouse monoclonal anti–band 3 (BRIC169), as described in “Materials and methods.” Immunoprecipitated protein was separated by SDS-PAGE and immunoblotted using rabbit polyclonal antibodies (“Materials and methods”) and a sheep anti–band 3.39Loading: ghost, RBC membrane protein; DC-IP, band 3 immunoprecipitate from deoxycholate-solubilized membrane proteins; DC solution, total deoxycholate-solubilized membrane proteins. Rabbit antibodies bound 2 bands in the DC-IP samples, at molecular weights of approximately 25 kDa and approximately 50 kDa (see panels AQP 1 and CD47, respectively), which probably results from cross-reactivity with the L and H chains of the large amount of mouse immunoglobulin in the immunoprecipitates.