Immunoblotting of Human Pepsinogens

Abstract To evaluate antisera raised against pepsinogen A and C, we applied the protein blotting technique to vertical polyacrylamide electrophoresis gels separating human gastric mucosal pepsinogens. After blotting in Veronal-TRIS buffer (pH 7.0) for one hour at 0.5 A (1.42 mA/cm 2 ), pepsinogens attached to nitrocellulose paper were detected by an indirect immunological method using goat antisera directed to human pepsinogen A or C followed by rabbit anti-goat IgG conjugated with Horse Radish peroxidase. Pre-immune serum showed no precipitation of the peroxidase substrate 3, 3′-diamino-benzidine, while anti-pepsinogen A stained band 1 to 5 of the electrophoresis pattern, and anti-pepsinogen C stained only band 6 and 7, as expected. By this method we could easily evaluate the specificity of goat antisera directed to the two immunological distinct groups of gastric mucosal pepsinogens.