Photoaffinity Labeling of RARs and Mapping of Labeled Sites by an Endoproteinase Combination Technique
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Photoaffinity labeling and mapping of the labeled site(s) are a superior and convenient methodology for initial structural investigation of ligand-receptor complexes. The method we describe in this chapter consists of five steps.
| 1. | Design and synthesis of a photoreactive probe for retinoic acid receptor (RAR)-labeling |
| 2. | Photoaffmlty labeling of RAR |
| 3. | Sequential digestion of the labeled RAR with endoproteinases |
| 4 | High-performance liquid chromatography (HPLC) analysis of the digests to determine the amino-acid sequence context of the labeled sites (endoproteinase combination technique). |
| 5. | Mapping of the labeled site(s) by comparison of the determined context with the known amino-acid sequence of the RAR</lt> |
