![]() Between the steps, various washes are done to increase the signal-to-noise ratio on the final, developed blot. Additional processing steps generate a signal at the position of the bound antibody. This membrane replica is treated with antibodies that specifically recognize a protein or epitope of interest. In a western blot procedure, proteins are first separated on an SDS-PAGE gel and then transferred to a membrane. If the molecule of interest is in the original mixture, it will “light" up and reveal itself. The secondary antibody is usually linked to an enzyme which, in the presence of the right reagent, catalyzes a reaction that produces a signal (color or light) indicating where the antibody is bound. The bound antibody can then be targeted by another antibody specific for the first antibody. For a protein, it would typically involve an antibody that specifically binds to the protein of interest. For DNA/RNA, that might be a complementary nucleic acid sequence that is labeled in some fashion (radioactivity or dye). Last, a visualizing agent specific for the molecule of interest in the mixture is added to the membrane. ![]() The membrane may be treated to covalently link the bands to the surface of the blot. The transfer can be accomplished by diffusion or by using an electrical current to move the molecules from the gel onto the membrane. This “blot", as it is called, has an imprint of the bands of nucleic acid or protein that were in the gel (see figure at left). Second, after the gel run is complete, the proteins or nucleic acids in the gel are transferred out of the gel onto a membrane/paper that physically binds to the molecules. The mixture could be DNA ( Southern Blot), RNA ( Northern Blot), or protein ( Western Blot) and the gel could be agarose (for DNA/RNA) or polyacrylamide (for protein). First, the mixture of molecules is separated by gel electrophoresis. \( \newcommand\)īlotting provides a means of identifying specific molecules out of a mixture.
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