[8 High Performance Affinity Beads for Identifying AntiNFcB Drug Receptors

By Masaki Hiramoto, Noriaki Shimizu, Takeyuki Nishi, Daisuke Shima, Shin Aizawa, Hirotoshi Tanaka, Mamoru Hatakeyama, Haruma Kawaguchi, and Hiroshi Handa

Introduction

Affinity purification is an established technique used to identify ligand-binding proteins1,2; however, its widespread use has been limited by the inefficiency and instability of conventional matrices. The unstable nature of conventional matrices narrows the spectrum of ligands that can be used. Moreover, nonspecific binding of proteins to the solid support has complicated identification of target proteins. Another difficulty is frequent low purification efficiency, which requires partial purification of the source material before affinity chromatography to improve results.

This chapter describes the preparation and use of affinity beads for identification of drug receptors. The drugs of interest are immobilized to the matrix, which consists of latex beads covalently coupled with spacers. The latex beads are composed of glycidylmethacrylate (GMA)-covered GMA-styrene (St) copolymer cores (SG beads) that were developed originally for the affinity purification of DNA-binding proteins.3-5 To reduce steric hindrance, divalent epoxide, that is, ethyleneglycol diglycidyl ether (EGDE), molecules are introduced as spacers after

1 P. Cuatrecasas, M. Wilchek, and C. B. Anfinsen, Proc. Natl. Acad. Sci. U.S.A. 61, 636 (1968).

2 P. Cuatrecasas and C. B. Anfinsen, Annu. Rev. Biochem. 40, 259 (1971).

3 H. Kawaguchi, A. Asai, Y. Ohtsuka, H. Watanabe, T. Wada, and H. Handa, Nucleic Acids Res. 17, 6229(1989).

4 Y. Inomata, H. Kawaguchi, M. Hiramoto, T. Wada, and H. Handa, Anal. Biochem. 206, 109 (1992).

5 T. Wada, H. Watanabe, H. Kawaguchi, and H. Handa, Methods Enzymol. 254, 595 (1995).

will not work in cell lines that fail to express either calcineurin or NF-AT. These problems can be circumvented by cotransfection with NF-AT and/or calcineurin expression plasmids. Electroporation has proved to be an easy and effective means of transfecting Jurkat cells with plasmid DNA, whereas other cell lines may require a different means of transfection (liposomes, retrovirus, CaCl2, etc.). This assay could be adapted to yield significant information about calcineurin activity in different human cell lines and provide a readout of h^C^-induced oxidative stress.

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