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Analyzing the regulation and function of ATM
journal contribution
posted on 2023-05-17, 13:03 authored by Lavin, MF, Scott, SP, Kozlov, S, Nuri GuvenNuri GuvenWe describe here the cloning of full-length ataxia-telangiectasia mutated (ATM) cDNA and characterization of its activity. Full-length ATM cDNA is cloned into an inducible EBV-based vector (pMEP4) and its expression analyzed in a stably transfected cell line. ATM protein induction is monitored by immunoblotting with antibodies against both ATM and a FLAG sequence tag in the recombinant protein. Extracts from irradiated cells are immunoprecipitated with anti-ATM antibodies, and protein kinase activity is measured using p53(1-44)-specific substrate or by immunoblotting extracts with an anti-phosphoserine 15 p53-specific antibody. Missense mutations affecting ATM kinase activity are detected using in vitro mutagenesis of ATM cDNA followed by the procedures outlined above.
History
Publication title
Methods in Molecular BiologyVolume
281Pagination
163-178ISSN
1064-3745Department/School
School of Pharmacy and PharmacologyPublisher
Humana Press, Inc.Place of publication
United StatesRights statement
Copyright 2004 Humanities Press Inc.Repository Status
- Restricted