Biotinylation of αENaC in kidneys and oocytes. Top: kidneys from rats on a low-Na diet were perfused with or without biotin. Lanes were loaded with 40 μg of total membrane protein or 25 μl of eluate from the neutravidin beads. Total membranes from whole kidney had three stained bands at ∼90, ∼65, and ∼30 kD. Only the 30-kD band was observed in eluates from neutravidin beads, but staining was similar in biotinylated and nonbiotinylated kidneys, indicating nonspecific binding to neutravidin beads. Bottom: The major peptide in ENaC-expressing oocytes is the full-length 90-kD form. Faint staining of material at this molecular mass was observed in the eluate of biotinylated but not of nonbiotinylated oocytes, showing that the protein can react with the biotin reagent. Similar results were obtained with three independent batches of oocytes. The 30-kD peptide could not be resolved due to nonspecific staining of an unidentified protein (not depicted).