Abstract
Various assays have been employed to study the nuclear receptor/cofactor interactions. Coimmunopre-cipitation protocols, both yeast and mammalian two-hybrid systems, and electrophoretic mobility shift/ supershift assays are all commonly used. One of the most useful assays for studying direct protein—protein interactions is the glutathione-S-transferase “pulldown” assay. We have developed a high-throughput version of this assay that utilizes a filter microplate to allow parallel processing of many samples, significantly reducing the time and reagents required for the assay and increasing the sensitivity of the assay for weaker protein—protein interactions.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Glass, C. K. (1996). Some new twists in the regulation of gene expression by thyroid hormone and retinoic acid receptors. J Endocrinol 150, 349–57.
Mangelsdorf, D. J.&Evans, R. M. (1995). The RXR heterodimers and orphan receptors. Cell 83, 841–50.
Glass, C. K.&Rosenfeld, M. G. (2000). The coregulator exchange in transcriptional functions of nuclear receptors. Genes Dev 14, 121–41.
Lazar, M. A. (2003). Nuclear receptor core-pressors. Nucl Recept Signal 1, e001.
Lee, J. W., Lee, Y. C., Na, S. Y., Jung, D. J.&Lee, S. K. (2001). Transcriptional coregulators of the nuclear receptor superfamily: coactivators and corepressors. Cell Mol Life Sci 58, 289–97.
Leo, C.&Chen, J. D. (2000). The SRC family of nuclear receptor coactivators. Gene 245, 1–11.
McKenna, N. J.&O'Malley, B. W. (2002). Minireview: nuclear receptor coactivators–an update. Endocrinology 143, 2461–5.
Ordentlich, P., Downes, M.&Evans, R. M. (2001). Corepressors and nuclear hormone receptor function. Curr Top Microbiol Immunol 254, 101–16.
Xu, J.&Li, Q. (2003). Review of the in vivo functions of the p160 steroid recep-tor coactivator family. Mol Endocrinol 17, 1681–92.
Rochette-Egly, C. (2003). Nuclear receptors: integration of multiple signalling pathways through phosphorylation. Cell Signal 15, 355–66.
Stallcup, M. R., Kim, J. H., Teyssier, C., Lee, Y. H., Ma, H.&Chen, D. (2003). The roles of protein-protein interactions and protein methylation in transcriptional activation by nuclear receptors and their coactivators. J Steroid Biochem Mol Biol 85, 139–45.
Weigel, N. L. (1996). Steroid hormone receptors and their regulation by phosphorylation. Biochem J 319 (Pt 3), 657–67.
Goodson, M. L., Farboud, B.&Privalsky, M. L. (2007). An improved high throughput protein-protein interaction assay for nuclear hormone receptors. Nucl Recept Signal 5, e002.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–5.
Wong, C., Sridhara, S., Bardwell, J. C.&Jakob, U. (2000). Heating greatly speeds Coomassie blue staining and destaining. Bio-techniques 28, 426–8, 430, 432.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Goodson, M.L., Farboud, B., Privalsky, M.L. (2009). High Throughput Analysis of Nuclear Receptor–Cofactor Interactions. In: McEwan, I.J. (eds) The Nuclear Receptor Superfamily. Methods in Molecular Biology™, vol 505. Humana Press. https://doi.org/10.1007/978-1-60327-575-0_9
Download citation
DOI: https://doi.org/10.1007/978-1-60327-575-0_9
Publisher Name: Humana Press
Print ISBN: 978-1-60327-574-3
Online ISBN: 978-1-60327-575-0
eBook Packages: Springer Protocols