Western Blotting (Immunoblotting) (PDF)
Introduction
The immunoblotting technique provides information about the presence, molecular weight, and/or quantity of an antigen by combining protein separation via gel electrophoresis with specific recognition of antigens by antibodies. Immunoblotting is useful when the antigen of interest is insoluble or readily degraded and cannot be easily immunoprecipitated. Since most gel electrophoresis procedures result in denaturation of the antigen, only polyclonal and monoclonal antibodies that recognize the denatured form of an antigen can be utilized in immunoblotting. To study proteins that are expressed at very low levels, it is
recommended that immunoprecipitation be followed by immunoblotting for more sensitive detection.