Antibody- Affinity Purification to Detect Interacting Proteins
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Affinity purification is a procedure based on the specific binding interactions between a ligand chemically coupled to a resin and a target molecule. A common application is the use of antibody as immobilized ligands. The purification of antigens by antibody-affinity chromatography is widely used to detect factors interacting with a protein of interest, and when coupled to mass spectrometry, it is a powerful approach for the identification of associated factors. In general, interacting proteins are isolated from cells expressing an epitope-tagged version of the protein of interest (1). Short peptides (tags) engineered at the amino- or carboxy-terminal end of proteins are quite useful for the purification of protein complexes through the use of tag-specific anti- body covalently coupled to a solid matrix. The use of tagged proteins is particularly effective for the isolation of native protein complexes from yeast cells, where these proteins can be expressed at their natural level (2, 3, 4). On the other hand, it is difficult to express engineered proteins at physiological levels in mammalian cells. Because overexpression may alter the network of protein interactions, the purification by affin- ity to an engineered epitope tag may not provide a faithful representation of the factors that are associated with the endogenous protein. To overcome this potential problem, many investigators choose to employ an affinity purification procedure that uses an antibody that recognizes the endogenous protein. This approach has been valuable in the identification of protein complexes involved in gene transcription (5- 6) and DNA repair (7).
KeywordsNuclear Extract Affinity Purification Sodium Metabisulfite Immobilize Ligand Wash Column
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