IMMUNOBLOTS
This immunological technique has gained great favor with both basic immunologysts and clinical immunologists over the past decade. Its beauty is its simplicity and the fact that one can compare different proteins, toxins and cellular products all at the same time and reach conclusions concerning their commonality or differences or purity. The procedure is relatively simple. The proteins to be studied are run on a standard SDS gel, the percentage of which depends on the known or estimated size of the protein: larger protein are run in 10% gels, while smaller proteins are run on 15% gels. The gels is then removed and the proteins in the gel are transferred by another electrical charge to a cellulose membrane. The membrane is treated overnight with a blocking buffer, washed, and then layered over the membrane with the antibody designed to pick up the binding to the protein in question. This incubation usually lasts one hour; following washes, the membrane is treated with a species-specific second antibody tagged to an enzyme and developed with an enzyme substrate to form a colored band.
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