Immunoprecipitation and Pull-Down of Nuclear Proteins

Splicing regulatory proteins usually function in larger complexes (see Chapters 3 Hertel, 5 Luhrmann, and 8 Smith). A protein complex can be purified using an antibody that is raised against a complex component. The antibody and the bound proteins are immobilized by protein A or G coupled to a solid support, such as Sepharose; proteins bound to the Sepharose may then be separated from unbound proteins by centrifugation. If no antiserum is available, the protein can be expressed as a tagged version in cells, and immunoprecipitated with available antisera against the tag. A related method is to immobilize a protein in vitro by using a protein tag that binds to a small compound, such as glutathione S-transferase, which in turn binds to glutathione that has been immobilized on a solid support. The immobilized protein can then be incubated with cell extracts or recombinant proteins to detect protein-protein interactions.