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Sci. STKE, 22 June 2004
Vol. 2004, Issue 238, p. pl10
[DOI: 10.1126/stke.2382004pl10]

PROTOCOLS

A Novel In Situ Assay for the Identification and Characterization of Soluble Nuclear Mobility Factors

Cem Elbi1, Dawn A. Walker1, Marcia Lewis2, Guillermo Romero2, William P. Sullivan3, David O. Toft3, Gordon L. Hager1*, and Donald B. DeFranco2*

1Laboratory of Receptor Biology and Gene Expression, Building 41, Room B602, National Cancer Institute, Bethesda, MD 20892–5055, USA.
2Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
3Department of Biochemistry and Molecular Biology, Mayo Graduate School, Rochester, MN 55905, USA.

Abstract: The development of green fluorescent protein (GFP) technology combined with live cell microscopy techniques have revealed the dynamic properties of GFP-tagged proteins in the nucleus. The mobility of a GFP-tagged protein can be assessed using a quantitative photobleaching technique, fluorescence recovery after photobleaching (FRAP) analysis. FRAP experiments demonstrate that many nuclear proteins are highly mobile within the nucleus. However, the factors within the nucleus that regulate this mobility are not known. This is partly due to an absence of protocols that can be used to identify such nuclear mobility factors. We developed a novel in situ assay that combines a biochemical permeabilization and extraction procedure with a quantitative FRAP technique, a method we used to uncover a new functional role for molecular chaperones in the nuclear mobility of steroid receptors. This assay can readily be adapted to identify and characterize other nuclear mobility factors.

*Corresponding authors: E-mail, hagerg{at}exchange.nih.gov (G.L.H.); dod1{at}pitt.edu (D.B.D.)

Citation: C. Elbi, D. A. Walker, M. Lewis, G. Romero, W. P. Sullivan, D. O. Toft, G. L. Hager, D. B. DeFranco, A Novel In Situ Assay for the Identification and Characterization of Soluble Nuclear Mobility Factors. Sci. STKE 2004, pl10 (2004).

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THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES:
The Ligand Binding Domain Controls Glucocorticoid Receptor Dynamics Independent of Ligand Release.
S. H. Meijsing, C. Elbi, H. F. Luecke, G. L. Hager, and K. R. Yamamoto (2007)
Mol. Cell. Biol. 27, 2442-2451
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