Western blot analysis can detect your protein of interest from a mixture of a great number of proteins. It 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). It can analyze any protein sample whether from cells 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.
In western blotting, proteins are electrophoresed in polyacrylamide gel, transferred onto a nitrocellulose or nylon membrane, and the protein bands are detected by their specific interaction with antibodies, lectins or some other compounds.
First step involves the separation of tissue. Then proteins are separated by gel electrophoresis from sample. If you are going to western blot for protein mass you can lyse in larger volumes If you are going to western blot a phospho-protein use phosphatase inhibitors. It will remove the phosphates from your proteins. If you are looking at protein-protein interactions use a less-stringent detergent such RIPA. ...Show more