False positive reactions can be identified when the immunoreactive protein band does not correspond in size to known viral proteins. The specificity of the western blot lies in its ability to demonstrate the molecular weights (sizes) of the proteins recognized by patient sera. The membrane is incubated with patient sera and the presence of patient antibodies is detected by subsequently incubating the membrane with labeled secondary antibodies. The proteins in a mixture are separated by size in the gel and are transferred from the gel to a solid (paper-like) substrate often called a membrane. Western blots are done by electrophoresis of antigen (i.e., purified virus) by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE). Western blots are labor intensive and expensive, but provide a method of confirming the identity of a reactive antigen. Susan Payne, in Viruses, 2017 Western Blots Before the next step (detection of bound antibody), nonbound primary antibody has to be washed out thoroughly. Due to the thickness of the membranes, incubations and washing steps are often longer than for solid phase immunoassays on nonporous surfaces such as microtiter plates. Only a small fraction of the antibody in the incubation mixture will be bound to the immobilized antigen, even if monospecific antibodies are used. As a rule of thumb, solutions of 1 μg ml −1 specific antibody incubated for several hours is a good starting point for optimizations. Hence, the signal ultimately generated will depend on the interplay of numerous factors such as affinity, concentration, duration of incubation, nonspecific binding, etc. In most respects, considerations given to solid phase immunoassays in microtiter plates also apply to western blotting. Western blotting may be performed with both polyclonal and monoclonal antibodies. Harry Towbin, in Encyclopedia of Immunology (Second Edition), 1998 Probing with specific antibodies It should equip the reader with the necessary knowledge to perform and interpret Western blot assays. In this chapter, we will (1) explain the principles underlying the Western blot assay (2) describe a basic protocol that can be adapted to specific requirements (3) provide examples of its use in biomedical research and diagnostic and (4) emphasize its key limitations. Many variations of this assay exist that can be summarized in several simple successive steps that makes Western blotting relatively easy to perform and troubleshoot. WB is a powerful technique to detect a single or a complex of proteins and to monitor changes in expression or posttranslational modifications. It utilizes simple principles to separate proteins according to their size and to bind them to a support allowing their detection with a specific antibody. Western blotting (WB) is a most common technique used to detect specific proteins in biological samples. Tomasz Gwozdz, Karel Dorey, in Basic Science Methods for Clinical Researchers, 2017 Abstract
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