Affinity labelling and cloning of steroid and thyroid hormone receptors / edited by H. Gronemeyer.

Date:
[1988]
Catalogue details

Licence: Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)

Credit: Affinity labelling and cloning of steroid and thyroid hormone receptors / edited by H. Gronemeyer. Source: Wellcome Collection.

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    Ch. 2] Glucocorticoid receptor 29 A. Preparation of samples 38 B. SDS-polyacrylamide gel electrophoresis of pH]Dex-Mes labeled receptors 38 C. Non-equilibrium pH gradient electrophoresis of pH]Dex-Mes labeled receptors 39 D. Isoelectric focusing of [^H]Dex-Mes labeled receptors on agarose gels 39 VIII. Covalent labeling of receptors in whole cells by Dex-Mex and localization of the covalent complexes 40 A. Labeling at 37°C 40 B. Labeling at 0°C 40 IX. Partial purification of unactivated affinity labeled receptors 41 A. (N64)2804 precipitation of [^H]Dex-Mes labeled receptors 41 B. Immunoadsorption of [^H]Dex-Mes labeled receptors 41 C. Affinity chromatography 42 X. Activation and DNA binding of affinity labeled receptors and purification of activated covalent complexes 42 A. Activation of [^H]Dex-Mes labeled receptors 42 B. Binding of [^H]Dex-Mes labeled receptors to DNA-cellulose column: and purification of activated complexes 43 C. Purification of Dex-Mes labeled receptors by 'double DNA-cellulose chromatography' 43 D. Binding to DNA-cellulose pellets 44 E. DNA competition binding assay 47 F. DNA filter binding and footprinting 47 XI. Proteolysis of unactivated, and activated, affinity labeled receptors. . .48 A. [^H]Dex-Mes labeled receptors 48 B. Proteolysis of photoaffinity labeled receptors 49 XII. Localization of modified amino acid(s) in affinity labeled receptors . . .49 A. [^H]Dex-Mes labeled receptors 49 References 52 I. INTRODUCTION Three general methods of affinity labeling exist: photoaffinity labeling, electrophilic affinity labehng, and chemoaffinity labeling (for review, see Simons and Thompson, 1982). The first two methods have been successful in yielding affinity labeled glucocorticoid receptors. However, most studies of the physical and biological properties of covalent receptor-steroid complexes have been conducted with elec¬ trophilic affinity labeled receptors. This chapter covers the most experimental techniques that have been employed to label glucocorticoid receptors covalently and to study the properties of the resultant covalent complexes.
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