LACK OF INTERSPECIES 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 protein s of different species. In the present study, we investigated the infl uence of mild protease digestion on the reactivity of nitrocellulose-b lotted proteins and microscopic sections with antibodies produced agai nst analogous proteins of different species. The proteins were partial ly purified, electrophoresed and blotted by standard procedures. They were then incubated with either trypsin or pepsin, at concentrations r anging from 0.5 to 80 mug/mL, for different time periods prior to reac tion with the first antibody. After incubation with the second antibod y, the bands were visualized by chemiluminescence. The following antib ody/antigen pairs produced no signal by conventional methods but resul ted in the appropriate bands following protease treatment: (i) monoclo nal antibody (Mab) to human keratin #18 / rat keratin; (ii) Mab to sta rfish extracellular matrix/mouse laminin; (iii) rabbit antiserum to hu man platelet myosin II/ratfish nonmuscle myosin II. In the latter syst em, protease treatment also revealed previously hidden epitopes in mic roscopic sections. Appropriate controls demonstrated that the antibodi es retained their specificity after the protease treatment of the prep arations. Optimal conditions varied and had to be defined for each pro tein under study. The results suggest that protease treatment of immun oblots reduces the lack of inter-species reactivity between antibodies and antigens.