Affinity crosslinking

One outcome of affinity labeling using the homologous series of bromoacetyl derivatives, which labeled different residues of the light and heavy chains, was that it enabled the first demonstration of affinity cross-linking by a reagent which contained two bromoacetyl groups and a DNP moiety. This reagent cross-linked the heavy and light chains at the correct positions. Affinity cross-linking is today a major tool in biology and immunology for the identification, localization and isolation of receptors. This is usually accomplished by using a radioactive ligand that is allowed to bind to its receptor, after which a general cross-linking reagent is applied. Alternatively, a ligand to which a photoreactive group has been attached can be used; after binding to the target membrane, light is applied which establishes a coval-ent bond at the site of binding. Affinity cross-linking allows the identification of the labeled receptor and determination of its size by gel electrophoresis. Because it makes use of a cross-linker which is separate from the ligand, it can be applied to large complex ligands such as growth factors and other proteins. Examples of the use of affinity cross-linking in the immune system are interleukin-2 and its receptor, Fc and receptor, major histocompatibility complex (MHC) class II molecules and nominal antigen peptides on antigen-presenting cells, and photoreactivc vinblastine and the 170 kDa protein from multidrug-resistant human cancer cells. Affinity cross-linking is mainly used to identify the molecular size and cellular localization of receptors. Its use, however, can be extended to study the ligand-binding site of the receptor, as has been shown for the epidermal growth factor receptor.

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