Stoichiometry of the oligomycin-sensitivity-conferring protein (OSCP) in the mitochondrial F0F1-ATPase determined by an immunoelectrotransfer blot technique

The ratio between the amount of oligomycin-sensitivity-conferring protein (OSCP) and the amount of the and β subunits of F₁-ATPase in the mitochondria has been determined by a method combining electrophoresis, electrotransfer and immunotitration with monoclonal antibodies. The peptides separated in SDS-polyacrylamide gel electrophoresis were blotted to nitrocellulose sheets by electrotransfer. The nitrocellulose sheets were incubated with ¹²⁵I-labelled purified monoclonal antibodies specific to various peptides. The ¹²⁵I-labelled immune complexes were located by immunodecoration using peroxidase-conjugated second antibodies and the blotted peptides were revealed with H₂O₂ and -naphthol. The amount of immune complex present on the nitrocellulose was determined by counting the radioactivity present on the spots. The amount of peptide blotted is directly proportional to the amount of protein loaded on the electrophoresis. By comparing standard curves made with the isolated proteins to the values obtained in the presence of various amounts of the membrane-protein complex, one can calculate the content of this peptide in the membrane. It was found that the mitochondrial membrane contains 2 mol of OSCP per mol of F₁.