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The streptavidin/biotinylated DNA/protein bound complex protocol for determining the association of c-JUN protein with NANOG promoter

SHAMS-NATERI, ABDOLRAHMAN; BABAEI-JADIDI, ROYA

Authors

ABDOLRAHMAN SHAMS-NATERI

ROYA BABAEI-JADIDI

Abstract

Chromatin immunoprecipitation (ChIP) is a widely used and pre-eminent technique for detecting the association of an individual protein or a particular protein complex with its specific DNA sequence(s) in vivo. Herein we introduce a novel and simple biotinylated-oligonucleotide-mediated ChIP method for testing specific binding of the c-JUN protein to the M1-DNA-regulatory element in the NANOG promoter. We prepared a 260-bp DNA PCR amplicon containing -300 bp to -59 bp, relative to the transcriptional start site of the human NANOG gene, which was transfected into mouse embryonic fibroblasts (MEF) containing wild-type (c-jun(+/+)) or knockout c-jun (c-jun(-/-)) alleles. Whole cells that were cross-linked using formaldehyde and protein-DNA interactions were immunoprecipitated using streptavidin-coupled Dynabeads. Protein-DNA cross-links were reversed during incubation at 95°C, and protein samples were visualized using SDS-PAGE electrophoresis and western blotting. This streptavidin/biotinylated DNA/protein-bound complex protocol can be used for detecting the interactions between multiple transcription factors and their DNA binding sites.

Journal Article Type Article
Publication Date May 17, 2013
Journal Current Protocols in Stem Cell Biology
Print ISSN 1941-7322
Electronic ISSN 1938-8969
Publisher Wiley
Peer Reviewed Peer Reviewed
Volume 25
Issue UNIT 1B.10
Article Number sc01b10s25
Pages 1-13
ISBN 9780470151808
Publisher URL https://currentprotocols.onlinelibrary.wiley.com/doi/10.1002/9780470151808.sc01b10s25
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