Validating Antibody Specificities for Immunohistochemistry by Protein Blotting

Optimized antibody reagents are important in research, and erratic antibody performance leads to variability in immunoassays. Specificity of antibodies binding the protein of interest is vital to obtain accurate results. Recommendations for validation and use of primary antibodies are unique to each type of immunoassay as the antibodies' performance is greatly affected by the assay context. Immunoblotting procedures have been used along with other important antibody-based detection methods like enzyme-linked immunosorbent assay and immunohistochemistry to confirm results in research and diagnostic testing. Specificity of antibodies employed for immunohistochemical studies is of critical importance. Therefore, the use of western blotting is imperative to address the specificity of antibodies with/without siRNA knockdown of proteins of interest or with the use of peptide inhibitors to inhibit the binding of specific antibodies to the target protein. In spite of its overall simplicity, western blotting or protein blotting is a powerful procedure for immunodetection of proteins, especially those that are of low abundance, following electrophoretic separation. The usefulness of this procedure stems from its ability to provide simultaneous resolution of multiple immunogenic antigens within a sample for detection by specific antibodies. Protein blotting has evolved greatly over the last few decades, and researchers have a variety of ways and means to carry out this procedure to validate antibodies for immunohistochemistry.