Unit

UNIT 1B.10 The Streptavidin/Biotinylated DNA/Protein Bound Complex Protocol for Determining the Association of c-JUN Protein with NANOG Promoter

  1. Elsayed E. Ibrahim1,2,
  2. Roya Babaei-Jadidi1,2,
  3. Abdolrahman S. Nateri1,2

Published Online: 1 MAY 2013

DOI: 10.1002/9780470151808.sc01b10s25

Current Protocols in Stem Cell Biology

Current Protocols in Stem Cell Biology

How to Cite

Ibrahim, E. E., Babaei-Jadidi, R. and Nateri, A. S. 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:B:1B.10:1B.10.1–1B.10.13.

Author Information

  1. 1

    Cancer Genetics and Stem Cell Group, Division of Pre-Clinical Oncology, University of Nottingham, Nottingham, United Kingdom

  2. 2

    Nottingham Digestive Diseases Centre, School of Clinical Sciences, University of Nottingham, Nottingham, Nottingham, United Kingdom

Publication History

  1. Published Online: 1 MAY 2013

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. Curr. Protoc. Stem Cell Biol. 25:1B.10.1-1B.10.13. © 2013 by John Wiley & Sons, Inc.

Keywords:

  • ChIP;
  • c-JUN;
  • NANOG;
  • streptavidin/biotin