An acetyltransferase assay for CREB-binding protein based on reverse phase-ultra-fast liquid chromatography of fluorescent histone H3 peptides.
Fiche publication
Date publication
octobre 2015
Journal
Analytical biochemistry
Auteurs
Membres identifiés du Cancéropôle Est :
Dr GUIDEZ Fabien
Tous les auteurs :
Duval R, Fritsch L, Bui LC, Berthelet J, Guidez F, Mathieu C, Dupret JM, Chomienne C, Ait-Si-Ali S, Rodrigues-Lima F
Lien Pubmed
Résumé
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.
Mots clés
CREB-binding protein, Fluorescent peptide, HPLC, Histone acetyltransferase, Histone methyltransferases, SETD2
Référence
Anal Biochem. 2015 10 1;486:35-7