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Western blot

A western blot is a laboratory method used to detect specific protein molecules from among a mixture of proteins. This mixture can include all of the proteins associated with a particular tissue or cell type. Western blots can also be used to evaluate the size of a protein of interest, and to measure the amount of protein expression. This procedure was named for its similarity to the previously invented method known as the Southern blot.

The first step in a western blot is to prepare the protein sample by mixing it with a detergent called sodium dodecyl sulfate, which makes the proteins unfold into linear chains and coats then with a negative charge. Next, the protein molecules are separated according to their sizes using a method called gel electrophoresis. Following separation, the proteins are transferred from the gel onto a blotting membrane. Although this step is what gives the technique the name "western blotting," the term is typically used to describe the entire procedure.

Once the transfer is complete, the membrane carries all of the protein bands originally on the gel. Next, the membrane goes through a treatment called blocking, which prevents any nonspecific reactions from occurring. The membrane is then incubated with an antibody called the primary antibody, which specifically binds to the protein of interest. Following incubation, any unbound primary antibody is washed away, and the membrane is incubated yet again, but this time with a secondary antibody that specifically recognizes and binds to the primary antibody. The secondary antibody is linked to a reporter enzyme that produces color or light, which allows it to be easily detected and imaged. These steps permit a specific protein to be detected from among a mixture of proteins.

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