Structure and specificity of DIDS-biotin reagent for band 3. (A) Fluorescent image of whole erythrocytes reacted with a subsaturating concentration of DIDS-biotin reagent (10 μM) and then labeled with Alexa Flour 598–derivatized streptavidin. (B) Image of unlabeled (control) whole erythrocytes that were similarly incubated with Alexa Fluor 598–derivatized streptavidin. (C) SDS-PAGE analysis of the specificity of DIDS-biotin reagent for band 3. Erythrocytes were either not reacted with DIDS-biotin reagent (lane 1) or reacted with 1 μM DIDS-biotin reagent (lane 2) before separation by SDS-PAGE. After electrophoretic transfer onto nitrocellulose, the molecular weights of DIDS-biotin–labeled polypeptides were determined by staining with streptavidin–horseradish peroxidase (1:1500). Lane 3 contains Coomassie blue–stained erythrocyte membranes, and lane 4 contains molecular weight markers. Vertical line(s) have been inserted to indicate a repositioned gel lane. (D) Structure of the DIDS-biotin reagent.