Western Blotting Technique in Biomedical Research
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Western Blot is the most important and powerful technique frequently used in laboratory research. It is used to identify specific proteins in biological samples isolated from cells or tissues. Similar to the Southern Blot for DNA and Northern Blot for RNA, the Western Blot procedures rely upon three key elements: the separation of protein mixtures by size utilizing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the efficient transfer of separated proteins from the gel to a nitrocellulose or polyvinylidene difluoride membrane, and identification of a target protein via conjugation with specific primary and enzyme-labeled secondary antibodies. This specificity of the antibody–antigen interaction enables a target protein to be identified in the midst of a complex protein mixture. Once detected, an appropriate substrate is then added to the enzyme, and together, they produce a detectable band visible on a blotting membrane, X-ray file, or imaging system. Western Blot is rapid, and simple, enabling easy to interpret, unique, and unambiguous results. Along with other immunoassays, Western Blot is routinely used in research and clinical settings.