Detection of protein blots using enzyme-linked second antibodies or protein a

Abstract

The immunodetection of a specific protein on a protein blot relies initially on the ability of a primary antibody to bind specifically to the protein of interest. This is simply achieved by washing the nitrocellulose paper in a dilute solution of the antibody. Before this step is carried out, however, unblocked protein binding sites on the nitrocellulose paper must be masked by washing with a general blocking solution (e.g., bovine serum albumin solution, dilute bovine serum, gelatin solution, hemoglobin solution, and so on). In this way, nonspecific binding of the antibody (which is itself a protein) to nitrocellulose is prevented, and any antibody that does bind to the paper will be doing so by a genuine antigen-antibody reaction alone. This antigen-antibody reaction must now be visualized. This can be achieved by washing the paper with an appropriately labeled ligand that binds specifically to the primary antibody. This ligand is generally of two possible types: 1. Protein A (which binds specifically to IgG molecules) either radiolabeled or linked to a marker enzyme (see below). 2. A second antibody (anti-IgG) that binds to the primary antibody. This second antibody is used linked to a marker enzyme. Alternatively this second antibody is linked to biotin. This biotinylated antibody is then treated with avidin linked to a marker enzyme (avidin binds with high specificity to biotin).