Heating of immunoglobulins for immunoblot analysis destroys variable region antigenicity.
The antigenicity of rabbit immunoglobulin heavy chain variable region allotypes was examined by immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The standard method of sample preparation resulted in a total loss of immunoreactivity with anti-allotype antibody due to heating of the samples prior to their electrophoresis. Thus, caution is warranted in interpreting results obtained from analysis of Ig when such proteins are heated before or during separation. In addition, we describe a method that permits not only separation of Ig heavy and light chains in SDS-PAGE but also preserves V region antigenicity.
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis; D006358:Hot Temperature; D007126:Immunoglobulin Allotypes / Q000032:analysis* / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000032:analysis* / Q000276:immunology; D011489:Protein Denaturation; D011817:Rabbits
D000939:Epitopes; D007126:Immunoglobulin Allotypes; D007135:Immunoglobulin Variable Region