IMMUNOBLOTTING

This chapter presents the technique of immunoblotting with emphasis on blotting of one-dimensional SDS gels onto nitrocellulose. Immunoblotting or Western blotting is a procedure in which a replica of a separating gel is produced by transferring proteins from a gel to a membrane such as nitrocellulose. This replica, or blot, is subsequently incubated or stained with antibodies. Immunoblotting provides information about an antigen, such as the subunit molecular weight of the antigens can be determined from its mobility in a sodium dodecyl sulfate (SDS) gel that is blotted. A blot can also give information about the purity of antibodies. Most types of gels, both one and two dimensional, can be blotted provided they are strong enough to withstand physical manipulation. Gels can be blotted either by submersed (wet) methods or by buffer-soaked filter paper (semidry) methods. Transfer can be accomplished either by passive diffusion or electrophoresis. The gel sandwich consists of the gel adjacent to the nitrocellulose with both enclosed by heavy filter paper. The paper and nitrocellulose are saturated with transfer solution to carry the electrical current. Nitrocellulose and other such membranes have a high affinity for proteins (and usually nucleic acids). The entire paper must be coated with protein, otherwise the primary antibody will bind everywhere nonspecifically. © 1993, Academic Press Inc.