title: The streptavidin/biotinylated DNA/protein bound complex protocol for determining the association of c-JUN protein with NANOG promoter.
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.
authors: Cancer Genetics and Stem Cell Group, Division of Pre-Clinical Oncology, University of Nottingham, Nottingham, United Kingdom.
authors: Elsayed E
authors: EE
authors: Ibrahim
authors: Roya
authors: R
authors: Babaei-Jadidi
authors: Abdolrahman S
authors: AS
authors: Nateri
mesh_terms: D000818:Animals; D019921:Biotinylation / Q000187:drug effects; D015153:Blotting, Western; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004622:Embryo, Mammalian / Q000166:cytology; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D051379:Mice; D000071317:Nanog Homeobox Protein; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011485:Protein Binding / Q000187:drug effects; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism*; D019809:Streptavidin / Q000378:metabolism*
chemical_list: D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D016755:Proto-Oncogene Proteins c-jun; D004247:DNA; D019809:Streptavidin