Affinity-based methods

Proteomics can make a key contribution in the identification of interacting protein partners and create a protein-protein interaction map of the cell (Lamond and Mann, 1997 Neubaer et ah, 1997 Blackstock and Weir, 1999 Link et ah, 1999). This would be of immense value to understanding the biology of the cell and can be further exploited for drug development. The simplest way to study such interaction is to purify the entire multiprotein complex by affinity-based methods. The members of the multiprotein complex can then be identified by mass spectrometry. 

The technique of choice in the detection and isolation of protein complexes is immunoprecipitation [15], whereas in some cases centrifugation or free-flow electrophoresis can be applied for the purification of cellular organelles. Immunoprecipitation permits the purification of specific proteins against which an antibody has been raised. Affinity-based methods have been developed for a variety of organelles. When apphed to the isolation of protein complexes, the antibody should target one of the proteins in the complex. The antibody-protein complex precipitates from solution. Alternatively, inunobilization of the primary antibody to agarose (beads) can be applied. Mrrlti-step procedttres have been developed as well. 

Selections and affinity-based methods have also been used to expand the scope of RNA-catalyzed reactions to other classes of reactions including isomerization, carbon-carbon bond forming, metalation, and alkylation reactions. For example, by exploiting the same notions of transition state theory that were used in the initial generation of catalytic antibodies, an RNA was isolated that catalyzes the isomerization of a bridged biphenyl substrate to its diastereomer Fig. 12) [89], In this case a library of 10 random RNA mole-... 

Furthermore, the activity/affinity-based methods are also suitable for identifying or annotating the functions of the gene product proteins as well as their drug ability (Barglow and Cravatt, 2007). 

The example also highlights that different affinity-based methods will be used at the different stages of the hit-to-lead process, which is very much determined by the required information content While the very sensitive NMR approach was used to identify weakly binding fragments, more potent compounds were assessed using the less-sensitive surface plasmon resonance (SPR) technology in order to drive the hit expansion process and to initiate and inform chemistry design. 

Fig.l. Three fundamentally different approaches for the application of affinity-based methods for hyphenated analytical systems. 

Nguyen, T. Joshi, N. S. Francis, M. B. An affinity-based method for the purification of fluorescently-labeled biomolecules. Bioconjugate Chem. 2006,17,869-872. 

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