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Steps in Western Blotting

 

western blot analysis can detect your protein of interest from a mixture of a great number of proteins. 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).

Summary: Western Blot Gives You Information on the:

Size of your Protein

Expression Amount of your Protein

Western blot analysis can analyze any protein sample whether from Cell s or tissues, but also can analyze recombinant proteins synthesized in vitro. 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. Antibodies are now easily obtainable from commercial sources, and you can purchase one for your protein of interest. If your protein is a novel protein, you must produce an antibody yourself or get a company to do it for you. 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). 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!

How Does Western Blotting Work?

See Diagram 1 below. First things first. Obtain a protein sample you want to analyze, such as cell samples. Lyse the cells to release protein contents. Run these on a gel which separates proteins on the basis of size. Then transfer these gel proteins onto a membrane using electricity. This membrane can then be used to probe for proteins of interest using a primary antibody.

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