Lack of inter-species reactivity between antigens and antibodies is overcome by protease treatment of western blots.
The analysis of proteins by Western blotting is frequently limited by the inability of antibodies to recognize antigenic epitopes in proteins of different species. In the present study, we investigated the influence of mild protease digestion on the reactivity of nitrocellulose-blotted proteins and microscopic sections with antibodies produced against analogous proteins of different species. The proteins were partially purified, electrophoresed and blotted by standard procedures. They were then incubated with either trypsin or pepsin, at concentrations ranging from 0.5 to 80 micrograms/mL, for different time periods prior to reaction with the first antibody. After incubation with the second antibody, the bands were visualized by chemiluminescence. The following antibody/antigen pairs produced no signal by conventional methods but resulted in the appropriate bands following protease treatment: (i) monoclonal antibody (Mab) to human keratin #18/rat keratin; (ii) Mab to starfish extracellular matrix/mouse laminin; (iii) rabbit antiserum to human platelet myosin II/ratfish nonmuscle myosin II. In the latter system, protease treatment also revealed previously hidden epitopes in microscopic sections. Appropriate controls demonstrated that the antibodies retained their specificity after the protease treatment of the preparations. Optimal conditions varied and had to be defined for each protein under study.(ABSTRACT TRUNCATED AT 250 WORDS)
Pan
J
J
Department of Anatomy, Faculty of Medicine, The University of British Columbia, Vancouver.
Pfeiffer
D C
DC
Reimer
C L
CL
Crawford
B J
BJ
Vogl
A W
AW
Auersperg
N
N
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D000941:Antigens / Q000276:immunology*; D015153:Blotting, Western; D002460:Cell Line; D010450:Endopeptidases / Q000494:pharmacology*; D005109:Extracellular Matrix / Q000276:immunology; D005399:Fishes / Q000276:immunology; D006801:Humans; D007633:Keratins / Q000276:immunology; D009218:Myosins / Q000276:immunology; D010434:Pepsin A / Q000494:pharmacology; D011506:Proteins / Q000032:analysis / Q000276:immunology*; D011817:Rabbits; D051381:Rats; D013045:Species Specificity; D013215:Starfish / Q000276:immunology; D014357:Trypsin / Q000494:pharmacology
D000906:Antibodies; D000911:Antibodies, Monoclonal; D000941:Antigens; D011506:Proteins; D007633:Keratins; D010450:Endopeptidases; D014357:Trypsin; D010434:Pepsin A; D009218:Myosins