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Lack of inter‐species reactivity between antigens and antibodies is overcome by protease treatment of Western blots
Author(s) -
Pan Jie,
Pfeiffer David C.,
Reimer Corinne L.,
Crawford Bruce J.,
Vogl A. Wayne,
Auersperg Nelly
Publication year - 1993
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.11501401142
Subject(s) - epitope , antibody , microbiology and biotechnology , monoclonal antibody , antigen , blot , protease , biology , antiserum , trypsin , keratin , tropomyosin , biochemistry , myosin , chemistry , enzyme , immunology , paleontology , gene
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 μg/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. The results suggest that protease treatment of immunoblots reduces the lack of inter‐species reactivity between antibodies and antigens.