Radiolabeled proteins, ligand binding

Different techniques have been applied to study the protein-protein, protein-ligand and, in particular, MIP-ligand interactions. They may serve to estimate or determine the binding constant and the number of independent binding sites (N) of a ligand-to-receptor (MIP or antibody) interaction. The range of affinity constants that can be calculated depends on the sensitivity of the assay and, in those cases where the separation of the bound and free species is a step of the assay, perturbation of the equilibria in the separation step will also be important [22]. Direct nonseparation techniques such as spectroscopic techniques (e.g., SPR or fluorescence polarization) can be used as well as indirect separation techniques such as radiolabeling [22]. 

A fluomicrosphere coated with protein will emit light only when the radiolabeled ligand binds to the protein when coming into proximity... 

Traditionally, the identification of protein targets of small molecules has relied on in vitro biochemical methods, such as photocross-linking, radiolabeled ligand binding, and affinity chromatography. Affinity chromatography is still a widely used method and can be used to identify targets present in any cell extract of choice. Therefore, it is, in principle, not restricted to an analysis of specific... 

To identify cell surface ligand-binding proteins, two different approaches to cross-linking may be employed. Figure 1 illustrates these two strategies which we have used successfully to identify the tissue-type plasminogen activator (tPA)-binding protein on hepatoma cells (Bu et al., 1992) either radiolabeled tPA crosslinked to unlabeled cells, or unlabeled tPA crosslinked to [ Sjmethionine metabolically labeled cells. Each of these two approaches is separately described in detail. 

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