Westerb Blotting (Handbook and Troubleshooting Guide)
Introduction The term “blotting” refers to the transfer of biological samples from a gel to a membrane and their subsequent detection on the surface of the membrane. Western blotting (also called immunoblotting because an antibody is used to specifically detect its antigen) was introduced by Towbin, et al. in 1979 and is now a routine technique for protein analysis. The specificity of the antibody-antigen interaction enables a single protein to be identified in the midst of a complex protein mixture. Western blotting is commonly used to positively identify a specific protein in a complex mixture and to obtain qualitative and semiquantitative data about that protein.
The first step in a Western blotting procedure is to separate the macromolecules using gel electrophoresis. Following electrophoresis, the separated molecules are transferred or blotted onto a second matrix, generally a nitrocellulose or polyvinylidene Difluoride (PVDF) membrane. Next, the membrane is blocked to prevent any nonspecific binding of antibodies to the surface of the membrane. The transferred protein is complexed with an enzyme-labeled antibody as a probe. An appropriate substrate is then added to the enzyme and together they produce a detectable product such as a chromogenic or fluorogenic precipitate on the membrane for colorimetric or fluorometric detection, respectively. The most sensitive detection methods use a chemiluminescent substrate that, when combined with the enzyme, produces light as a byproduct. The light output can be captured using film, a CCD camera or a phosphorimager that is designed for chemiluminescent detection. Whatever substrate is used, the intensity of the signal should correlate with the abundance of the antigen on the blotting membrane.
Detailed procedures for detection of a Western blot vary widely. One common variation involves direct vs. indirect detection as shown in Figure 1. With the direct detection method, the primary antibody that is used to detect an antigen on the blot is also labeled with an enzyme or fluorescent dye. This detection method is not widely used as most researchers prefer the indirect detection method for a variety of reasons.
In the indirect detection method, a primary antibody is added first to bind to the antigen. This is followed by a labeled secondary antibody that is directed against the primary antibody. Labels include biotin, fluorescent probes such as fluorescein or rhodamine, and enzyme conjugates such as horseradish peroxidase or alkaline phosphatase. The indirect method offers many advantages over the direct method.