Absence of biotin labeling of intracellular membrane proteins. Kidneys were perfused with or without biotin. Lanes were loaded with 15 μg of total membrane protein or 20 μl of eluate from the neutravidin beads. Duplicate adjacent lanes represent separate material from each of two animals for each condition. The upper blot was stained with anti-golgin; the full length protein corresponds to the doublet of high apparent molecular mass. No staining of the eluates could be detected either with or without biotin. The lower blot was stained with anti-calnexin, which gave a very strong signal with the total membrane pool. Similar staining was observed with the eluates from kidneys perfused with or without biotin, indicating the absence of biotinylation of this protein. In these and subsequent blots, numbers to the right correspond to the positions of molecular mass standards in kD.