Affinity capture and identification of drug targets

Before the era of artificially recombinant DNA, affinity chromatography emerged as a potentially highly selective approach to protein purification [13]. It revived a laborious art based on selective precipitations and a limited range of chromatographic media. 

Successful affinity chromatography requires that the protein interact with an immobilized ligand tightly enough to be captured from solution, 

Nonspecific protein binding to the solid phase complicates the method and is a selective pressure driving its evolution. The adaptive response has been the development of intrinsically comparative methods in which specific binding to an immobilized ligand is blocked in one out of two otherwise identical samples. When the respective protein components of the samples are compared, specifically bound proteins are present in one but severely depleted in the other. To allow relative quantitation, the two samples can be made isotopically distinct by a chemical or metabolic process and then mixed for an analytical step that avoids intersample variability [15]. 

Bead-bound PF-4540124 (2) was incubated with the soluble fraction of a mouse lung homogenate in the presence and absence of 1 (100 J.M). The type of protein captured was restricted by having 1 mM ADP and GDP in both samples. The beads were pelleted and washed before being treated (both samples) with 1 (100 iM) to elute specifically bound proteins. 

An important factor in all these experiments is the choice of bead used to immobilize the probe. Biochemists have considered cross-linked agarose beads to be exceptionally hydrophilic with a low tendency to bind proteins nonspecifically, and these beads have the further attraction of being commercially available in activated forms (succinimidyl esters, epoxides, and maleimides, for example). However, early trials of bead-based chemical proteomics have shown that many proteins in mammalian cell lysates bind tenaciously to agarose beads. This was unimportant in many studies in which protein-protein interactions were detected by coimmunoprecipitation with immunochemical 

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