User:Farooqak10/Western blot

western blot technique uses three elements to achieve its task of separating a specific protein from a complex: separation by size, transfer of protein to a solid support, and marking target protein using a primary and secondary antibody to visualize. A synthetic or animal-derived antibody (known as the primary antibody) is created that recognises and binds to a specific target protein. The electrophoresis membrane is washed in a solution containing the primary antibody, before excess antibody is washed off. A secondary antibody is added which recognises and binds to the primary antibody. The secondary antibody is visualised through various methods such as staining, immunofluorescence, and radioactivity, allowing indirect detection of the specific target protein.

To make the proteins accessible to antibody detection, they are moved from within the gel onto a membrane, a solid support, is an essential part of the process. There are two types of membrane: nitrocellulose (NC) or polyvinylidene difluoride (PVDF). NC membrane has high affinity for protein and its retention abilities. however, NC is brittle, and does not allow the blot to be used for re-probing. whereas, PVDG membrane allows the blot to be re-probed.

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=== References Mahmood, T., & Yang, P.-C. (2012). Western blot: Technique, theory, and trouble shooting. North American Journal of Medical Sciences, 4(9), 429. https://link.gale.com/apps/doc/A302531260/AONE?u=cuny_hunter&sid=AONE&xid=d331f064 ===