Proteins surface affinity

Once the protein is modified to contain an alkynyl group at its C-terminal it can be used to covalently link to its click chemistry reactant partner, an azide on the surface of an array. Other azido molecules also can be conjugated with an alkyne-protein to facilitate the detection or capture of the protein using affinity techniques. For instance, an azido-fluorescein reagent can be used to detect fluorescently the expressed protein in complex samples or an azido-biotin... 

A number of other issues must now be addressed including determining if the IgG-protein A affinity is sufficiently high to keep the antibody associated with the virus in vivo. The full potential of this approach will also require more detailed characterization of surface markers uniquely associated with different cell types. However, the approach exemplifies the types of technical innovation now being introduced that will make second-generation vectors more suited to their role in gene therapy. 

Affinity chromatography and related techniques (e.g., thiol chromatography and IMAC) are widely used for preparative isolation because they enable a single protein or class of proteins to be selectively purified from very complex mixtures. They may be occasionally used as analytical tools. For example, protein A affinity chromatography has been used for quantitative analysis of immunoglobulins in ascites fluid.45 Information about surface-accessible histidine and phosphate groups may be obtained using IMAC. 

Zamora, I., Oprea, T., Cruciani, G., Pastor, M., and Ungell, A.-L. Surface descriptors for protein-ligand affinity prediction. /. Med. Chem. 2003, 46, 25-33. 

For example, our preliminary MALDI MS studies employed a somewhat unconventional sample preparation approach in which the protein deposition step preceded the matrix deposition step. (In the conventional approach, the protein is co-deposited with the MALDI matrix solution.). .. Thus, in the first/next/colleclively group of experiments, we will explore different sample preparation methods to determine if other protocols also lead to an inverse relationship between surface-protein binding affinity and the MALDI ion signal. 

These studies will be followed with experiments that examine the influence of the matrix solvent on the inverse surface-protein binding affinity/MALDI ion signal relationship. 

Briesewitz, R., Ray, G. T., Wandless, T. J., and Crabtree, G. R. (1999). Affinity modulation of small-molecule ligands by borrowing endogenous protein surfaces. Proc. Natl. Acad. Sci. U.S.A. 96, 1953-1958. 

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