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

Elsayed E. Ibrahim1, Roya Babaei‐Jadidi1, Abdolrahman S. Nateri1

1 Nottingham Digestive Diseases Centre, School of Clinical Sciences, University of Nottingham, Nottingham, Nottingham, United Kingdom
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1B.10
DOI:  10.1002/9780470151808.sc01b10s25
Online Posting Date:  May, 2013
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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 (cjun+/+) 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

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Table of Contents

  • Introduction
  • Basic Protocol 1: Chromatin Immunoprecipitation (ChIP): Cell Preparation and Cross‐Linking
  • Basic Protocol 2: Immunoprecipitation (IP) and Reversal Cross‐Linking
  • Support Protocol 1: Designing Oligonucleotides and NANOG Promoter Amplification
  • Support Protocol 2: Detection of the NANOG Promoter–Associated Protein (e.g., c‐JUN) Using Western Blotting
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Chromatin Immunoprecipitation (ChIP): Cell Preparation and Cross‐Linking

  • Mouse embryonic fibroblasts (MEFs; c‐jun+/+ and c‐jun−/−; gift from Dr. Axel Behrens, Cancer Research UK–London Research Institute)
  • 1% (w/v) gelatin (Sigma, cat. no. G7765)
  • Phosphate‐buffered saline (PBS; Sigma, cat. no. P4417; one tablet dissolved in 200 ml of deionized H 2O yields 0.01 M phosphate buffer/0.0027 M potassium chloride/0.137 M sodium chloride, pH 7.4, at 25°C)
  • Dulbecco's modified Eagle medium (DMEM; Sigma, cat. no. D5671)
  • Complete DMEM growth medium (see recipe)
  • NANOG‐promoter PCR product ( protocol 3)
  • Opti‐MEM I reduced‐serum medium (Invitrogen, cat. no. 31985‐047)
  • Lipofectamine 2000 reagent (Invitrogen, cat. no. 11668‐019)
  • 11% (v/v) formaldehyde solution (see recipe)2.5 M glycine (Sigma, cat. no. G8898)
  • RIPA lysis buffer (see recipe)
  • 10‐cm tissue culture plates
  • Rubber scraper
  • Centrifuge
NOTE: All incubations are performed in a 37°C, 5% CO 2 humidified incubator, unless otherwise noted.NOTE: All reagents and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.

Basic Protocol 2: Immunoprecipitation (IP) and Reversal Cross‐Linking

  • Dynabeads M‐280 Streptavidin (Invitrogen, cat. no. 112.05D)
  • Binding & washing (B & W) buffer (see recipe)
  • DNA‐protein extract ( protocol 1)
  • Dulbecco's phosphate‐buffered saline (DPBS; Sigma, cat. no. D8662) containing 0.1% (v/v) NP‐40 and 1× fresh protease inhibitor cocktail (Sigma, cat. no. P2714)
  • 5× loading buffer (see recipe)
  • DynaMag‐2 magnet (Invitrogen, cat. no. 123‐21D)
  • Shaking thermomixer

Support Protocol 1: Designing Oligonucleotides and NANOG Promoter Amplification

  • Biotinylated and non‐biotinylated oligonucleotides corresponding to NANOG promoter (nucleotides –300 to –59; see above)
  • Nuclease‐free H 2O
  • Human embryonic stem cells (hESCs; unit 1.5)
  • Phosphate‐buffered saline (PBS; Sigma, cat. no. D8537)
  • DirectPCR lysis reagent (Viagen Biotech, cat. no. 402E,
  • Proteinase K (Sigma, cat. no. P6556)
  • Extracted genomic template DNA
  • 10× PCR reaction buffer (Qiagen, cat. no. 201223)
  • 0.25 mM dNTP mix (Qiagen, cat. no. 201913)
  • 5 U/µl Taq DNA polymerase (Qiagen, cat. no. 201205)
  • Agarose powder (Eurogenetic, cat. no. EP‐0010‐05)
  • Tris‐acetate‐EDTA (TAE) buffer, pH 8.0 (Sigma, cat. no. T9650)
  • Ethidium bromide (EtBr; Sigma, cat. no. E1385)
  • Illustra GFX PCR DNA and Gel Band purification kit (GE Healthcare, cat. no. 28‐9034‐70)
  • Shaking Thermomixer (Fisher, cat. no. BLD‐455‐010C)
  • 0.2‐ml PCR tubes (Qiagen)
  • Thermal cycler (Applied Biosystems)

Support Protocol 2: Detection of the NANOG Promoter–Associated Protein (e.g., c‐JUN) Using Western Blotting

  • 1× Tris/glycine/SDS (TGS) running buffer (Bio‐Rad, cat. no. 161‐0772)
  • Semi‐dry transfer buffer (see recipe)
  • Dried milk (GE Healthcare, cat. no. CPK1075)
  • Tris‐buffered saline (TBS; see recipe)
  • Tween 20 (Sigma, cat. no. 1379)
  • Primary antibody: c‐JUN antibody (from rabbit; Santa Cruz Biotechnology, cat. no. sc‐1694)
  • Secondary antibody: directed against rabbit Ig
  • Polyvinylidene difluoride (PVDF) membrane (GE Healthcare, cat. no. RPN303F)
  • Semi‐dry electrophoretic transfer cell (Bio‐Rad)
  • Platform rotator
  • ECL Advanced Western blotting detection kit (Amersham, cat. no. RPN2135)
  • FluorChem FC2 imaging system (Alpha Innotech)
  • Additional reagents and equipment for SDS‐PAGE (Gallagher, ) and protein blotting (Gallagher et al., 2008)
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