Nonradioactive Photoaffinity Labeling of Steroid Receptors Using Western Blot Detection System
Photoaffinity labeling is the process of covalently crosslinking a photoactive ligand, which can be detected postcoupling to a receptor or binding protein. This technique has proven useful in the identification of novel proteins or to further study and characterize known proteins. Photoaffinity labeling of novel receptors and binding proteins can provide information about biochemical properties of the target protein by allowing it to be traced through various separation schemes. For example, visualizing a labeled protein following one- or two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) can provide information about its apparent molecular weight (mol wt) and isoelectric point (1, 2). Knowledge of these biochemical characteristics, and others, can be useful in further design of purification strategies. Photoaffinity labeling of known receptors or binding proteins can be used to study important residues in the target protein’s binding site. For example, photoaffinity labeling, followed by an enzymatic digest and sequencing of the labeled peptide(s), allows the identification of specific labeled residues in or near the binding pocket (3) Other studies might also use photoaffinity labels to induce a chronic activation or inactivation of a receptor by crosslinking the ligand to the binding site.
KeywordsChemiluminescent Detection System Radioligand Binding Assay Photoaffinity Label Nonspecific Label Western Blot Procedure
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