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9.11: Blotting

Blotting provides a means of identifying specific molecules out of a mixture. It employs three main steps. First, the mixture of molecules is separated by gel electrophoresis. The mixture could be DNA (Southern Blot), RNA (Nothern Blot), or protein (Western Blot) and the gel could be agarose (for DNA/RNA) or polyacrylamide (for protein). 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. 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). The transfer can be accomplished by diffusion or by using an electrical current to move the molecules from the gel onto the membrane. The membrane may be treated to covalently link the bands to the surface of the blot. Last, a visualizing agent specific for the molecule of interest in the mixture is added to the membrane. For DNA/RNA, that might be a complementary nucleic acid sequence that is labeled in some fashion (radioactivity or dye). For a protein, it would typically involve an antibody that specifically binds to the protein of interest. The bound antibody can then be targeted by another antibody specific for the first antibody. 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. If the molecule of interest is in the original mixture, it will “light" up and reveal itself.

Figure 9.11.1: Northern blotting procedure

Contributors

Dr. Kevin Ahern and Dr. Indira Rajagopal (Oregon State University)