Production of monospecific antibodies to a low-abundance hepatic membrane protein using nitrocellulose immobilized protein as antigen.
Membrane proteins from primary cultures of rat hepatocytes were separated by two-dimensional polyacrylamide gel electrophoresis. The proteins were transferred to nitrocellulose paper which was then dissolved in dimethyl sulfoxide and this mixture was used as a primary immunogen in rabbits. Subsequent immunizations were performed using nonsolubilized protein immobilized on nitrocellulose paper. A monospecific polyclonal antibody was generated against a specific mitochondrial membrane protein (MP-73) for which de novo synthesis appeared to be induced by amino acid starvation of the hepatocytes. A minimum of 15-20 micrograms of protein antigen was required to elicit significant antibody production. Serum antibody titer was sufficient to allow detection of MP-73 at a serum dilution of 1:2000.
Chiles
T C
TC
O'Brien
T W
TW
Kilberg
M S
MS
D000818:Animals; D000917:Antibody Formation; D000918:Antibody Specificity; D000941:Antigens / Q000032:analysis; D003101:Collodion; D004591:Electrophoresis, Polyacrylamide Gel; D007158:Immunologic Techniques; D008099:Liver / Q000032:analysis*; D008297:Male; D008565:Membrane Proteins / Q000276:immunology* / Q000302:isolation & purification; D051381:Rats; D011919:Rats, Inbred Strains
D000941:Antigens; D008565:Membrane Proteins; D003101:Collodion