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Development of a Binding Assay for p53/HDM2 by Using Homogeneous Time-Resolved Fluorescence by Stefanie A. Kane; Catherine A. Fleener; Yun S. Zhang; Lenora J. Davis; Amy L. Musselman; Pearl S. Huang is a Biochemistry, Genetics and Molecular Biology article available to read on EtoBox.

What is Development of a Binding Assay for p53/HDM2 by Using Homogeneous Time-Resolved Fluorescence about?

The p53 tumor suppressor protein is activated and stabilized in response to DNA damage, resulting in cell cycle arrest or apoptosis. HMD2 is a negative regulator of p53. Binding of p53 by HDM2 traffics p53 from the nucleus to the cytoplasm where it is recognized and targeted for ubiquitin-mediated degradation (D. A. Freedman, L. Wu, and A. J. Levine, 1999, Cell. Mol. Life Sci. 55, 96-107). Several reports have suggested that disruption of this complex in normal cells results in p53 signaling (V. Böttger, A. Böttger, A. Sparks, W.-L. Liu, S. F. Howard, and D. P. Lane, 1997, Curr. Biol. 7, 860-869; C. Wasylyk, R. Salvi, M. Argentini, C. Dureuil, I. Delumeau, J. Abecassis, L. Debussche, and B. Wasylyk, 1999, Oncogene 18, 1921-1934). A homogeneous time-resolved fluorescence (HTRF) assay has been developed to monitor p53/HDM2 binding. This assay employs a site-specific biotinylated p53 protein, a GST-fused HDM2 protein, and two fluorophore-conjugated detection reagents, streptavidin-XL665 and europium cryptate-labeled anti-GST antibody ¿Eu(K)-anti-GST. Binding of p53 to HDM2 brings the fluorophores into close proximity, allowing fluorescence resonance energy transfer to occur. Developme

Who reads Development of a Binding Assay for p53/HDM2 by Using Homogeneous Time-Resolved Fluorescence?

Author

Stefanie A. Kane; Catherine A. Fleener; Yun S. Zhang; Lenora J. Davis; Amy L. Musselman; Pearl S. Huang

Publisher

Elsevier Science; Elsevier ; Elsevier Inc.; Elsevier BV (ISSN 0003-2697)

Published

2000

Language

EN

Field

Biochemistry, Genetics and Molecular Biology (Life Sciences)