![]() Western blot relies on the primary antibody to detect this protein from the thousands of proteins on your membrane and previously on your gel! (a cell can contain 30,000 different proteins – and these same proteins can even be altered giving you over 300,000 different proteins!). This membrane can then be used to probe for proteins of interest using a primary antibody.Ī Good Antibody to Detect your Protein of Interest Then transfer these gel proteins onto a membrane using electricity. Run these on a gel which separates proteins on the basis of size. Lyse the cells to release protein contents. Obtain a protein sample you want to analyze, such as cell samples. Antibodies specific to your protein are vital to western blotting as they are able to bind specifically to your protein of interest instead of the thousands of proteins on your western blot! In this case you will need at least a small amount of your protein either purified from cell extracts or made as a recombinant (ie in vitro or in a recombinant protein expression system). ![]() If your protein is a novel protein, you must produce an antibody yourself or get a company to do it for you. Antibodies are now easily obtainable from commercial sources, and you can purchase one for your protein of interest. Western blot is dependent on the quality of antibody you use to probe for your protein of interest, and how specific it is for this protein. Western blot analysis can analyze any protein sample whether from cells or tissues, but also can analyze recombinant proteins synthesized in vitro. Summary: Western Blot Gives You Information on the: Western blotting can give you information about the size of your protein (with comparison to a size marker or ladder in kDa), and also give you information on protein expression (with comparison to a control such as untreated sample or another cell type or tissue). ![]() Western blot analysis can detect your protein of interest from a mixture of a great number of proteins. ![]() Western Blot Definition: Western blotting is a technique used to identify and locate proteins based on their ability to bind to specific antibodies. ![]()
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