The Streptavidin/Biotinylated DNA/Protein Bound Complex Protocol for Determining the Association of c‐JUN Protein with NANOG Promoter

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.