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

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.

Citation

SHAMS-NATERI, A., & BABAEI-JADIDI, R. (2013). The streptavidin/biotinylated DNA/protein bound complex protocol for determining the association of c-JUN protein with NANOG promoter. Current Protocols in Stem Cell Biology, 25(UNIT 1B.10), 1-13

• Journal Article Type: Article
• Acceptance Date: Jan 28, 2013
• Online Publication Date: Jun 15, 2007
• Publication Date: May 17, 2013
• Deposit Date: Dec 13, 2018
• 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
• Public URL: https://nottingham-repository.worktribe.com/output/1410392
• Publisher URL: https://currentprotocols.onlinelibrary.wiley.com/doi/10.1002/9780470151808.sc01b10s25