Band 3 Neapolis protein analysis. (A) Immunoblotting of tryptic fragments of band 3. Red-cell membranes were digested by trypsin, and band 3 in the supernatant fraction was purified using an immunoaffinity column. The fraction eluted with 1 M glycine, pH 3.0 was analyzed by immunoblotting, using antibodies directed against band 3. The top panel shows the results from a short exposure of film, while the bottom panel represents a longer exposure. (B) The potential ATG start sites in exon 3. (C) Mass spectrometric analysis of normal and mutant band 3. Each of the derived signals was characterized on the basis of its molecular mass and protease specificity. Asterisks indicate peptides generated from endoprotease AspN autoproteolysis. (D) Immunoblotting of band 3. Equivalent amounts of red-cell membranes were analyzed by immunoblotting, using monoclonal antibodies directed against N-terminal amino acids (clone CDB3). Note complete absence of the band 3 epitope in red-cell membranes of the proband.