The immunoblotting (Western blotting) 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.
The specificity of antibodies to identify a single protein is utilized. Immunoblotting is a good choice when small quantities of sample are available, as it can detect picogram amounts of protein with the right antibody. It 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 (SDS-PAGE), 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.