Elsevier

Analytical Biochemistry

Volume 486, 1 October 2015, Pages 35-37
Analytical Biochemistry

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An acetyltransferase assay for CREB-binding protein based on reverse phase–ultra-fast liquid chromatography of fluorescent histone H3 peptides

https://doi.org/10.1016/j.ab.2015.06.024Get rights and content

Abstract

CREB-binding protein (CBP) is a lysine acetyltransferase that regulates transcription by acetylating histone and non-histone substrates. Defects in CBP activity are associated with hematologic malignancies, neurodisorders, and congenital malformations. Sensitive and quantitative enzymatic assays are essential to better characterize the pathophysiological features of CBP. We describe a sensitive nonradioactive method to measure purified and immunopurified cellular CBP enzymatic activity through rapid reverse phase–ultra-fast liquid chromatography (RP–UFLC) analysis of fluorescent histone H3 peptide substrates. The applicability and biological relevance of the assay are supported by kinetic, inhibition, and immunoprecipitation studies. More broadly, this approach could be easily adapted to assay other lysine acetyltransferases or methyltransferases.

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Acknowledgments

This work was supported by the Institut National du Cancer (INCa, 2012-1-PL-BIO 2012, coordinated by S. Ait-Si-Ali) and the ITMO Cancer (plan Cancer-Environnement 2013, coordinated by C. Chomienne and F. Rodrigues-Lima). R.D. and C.M. are supported by PhD fellowships from the Région Ile-de-France and Université Paris Diderot, respectively. UFLC analyses were done on the platform “Bioprofiler” (Unité de Biologie Fonctionnelle et Adaptative, BFA).

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