Immobilized metal ion affinity chromatography (IMAC) has proved useful in isolating proteins present in trace amounts from complex mixtures. The objective of this award is to characterize and map several IMA-adsorbents and to use the results of these studies to formulate and test protocols for the isolation of proteins from crude mixtures using cascade mode multi-affinity chromatography (CASMAC). The first goal is to characterize new gels with regard to chelating agents, metal ions and chromatography conditions. Among other methods, adsorption isotherms and NMR will be used. Secondly, these gels will be mapped using retention studies of amino acids, and model peptides and proteins. Thirdly, the investigators plan to develop and test protocols that allow fractionation of complex protein mixtures using CASMAC. It is anticipated that this work will result in making adsorbents available to the scientific community for both IMAC and CASMAC that offer incremental affinities for proteins and peptides, and that are specifically targeted for interaction with different amino acid side groups. Further the investigators plan to develop a rational approach using a series of affinity techniques to separate complex protein mixtures effectively.
