Antibody Validation by Western Blotting

Validation of antibodies is an integral part of translational research, particularly for biomarker discovery. Validation is essential to show the specificity of the reagent (antibody) and to confirm the identity of the protein biomarker, prior to implementing the biomarker in clinical studies.Antibody validation is the procedure in which a single antibody is thoroughly assayed for sensitivity and specificity. Although a plethora of commercial antibodies exist, antibody specificity must be thoroughly demonstrated using a complex biological sample, rather than a recombinant protein, prior to use in clinical translational research. In the simplest iteration, antibody specificity is determined by the presence of a single band in a complex biological sample, at the expected molecular weight, on a western blot.Numerous western blotting procedures are available, spanning the spectrum of single blots to multiplex blots, with images and quantitation generated by manual or automated systems. The basic principles of western blotting are (a) separation of protein mixtures by gel electrophoresis, (b) transfer of the proteins to a blot, (c) probing the blot for a protein or proteins of interest, and (d) subsequent detection of the protein by chemiluminescent, fluorescent, or colorimetric methods. This chapter focuses on the chemiluminescent detection of proteins using a manual western blotting system and a vacuum-enhanced detection system (SNAP i.d.™, Millipore).