Fig. 2. Detection of bound anti-HA antibody to tagged subunits in intact vacuoles. (A) Wild type (WT), (B) Vma7p-HA, (C) Vma10p-HA, (D) Vma16p-HA. Approx. 400 µg of vacuoles were diluted and incubated for 30 min at room temperature with 50 µl of monoclonal anti-HA antibody. After two washes with the dilution buffer to remove the unbound anti-HA antibody, the vacuoles were solubilized by detergent, loaded on top of a 20%–50% glycerol density gradient and centrifuged at 435 500 g for 13 h. 12 fractions were collected from the bottom of the gradient. Protein samples from all fractions were analyzed by western blot. Lanes a were decorated with HRP-conjugated sheep anti-mouse Ig antibody; lanes b, anti-HA antibody, lanes c, anti-Vma5p antibody, lanes d, anti-Vma1p antibody and lanes e, anti-Vph1p antibody (see Materials and methods).