Western blotting for protein analysis from "summary" of Molecular Cloning by Joseph Sambrook,David William Russell
Western blotting is a widely used technique for detecting specific proteins in a complex mixture of proteins extracted from cells or tissues. The technique involves separating the proteins based on their size using gel electrophoresis and then transferring them onto a membrane for detection. The first step in Western blotting is to separate the proteins by size using SDS-PAGE, a technique that involves denaturing the proteins with a detergent called sodium dodecyl sulfate (SDS) and running them through a polyacrylamide gel. The proteins migrate through the gel at different rates based on their size, with smaller proteins moving faster than larger ones. After the proteins have been separated, they are transferred from the gel onto a membrane by a process known as electroblotting. In this step, an electric current is applied to the gel, causing the proteins to move out of the gel and onto the membrane, where they adhere based on their size and charge. Once the proteins are transferred onto the membrane, they can be detected using specific antibodies that bind to the target protein. These antibodies are typically labeled with a detection reagent, such as an enzyme or a fluorophore, which produces a signal that can be visualized using various imaging techniques. Western blotting is a powerful tool for analyzing protein expression levels, post-translational modifications, and protein-protein interactions. It is commonly used in research labs and clinical settings to study a wide range of biological processes, including disease mechanisms, drug discovery, and biomarker identification.
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