- Home
- Protocols
-
Yeast Transcription Factor Chromatin Immunoprecipitation
Published: Jul 5, 2011 DOI: 10.21769/BioProtoc.92 Views: 13368
PDF 
Ask a question
Abstract
This ChIP protocol was developed and improved over the years by various researchers in the Snyder lab, Stanford University, especially Anthony Borneman and Christopher Yellman. I have used this method to successfully map the genome-wide binding of transcription factors Ste12. The ChIPed DNA is suitable for downstream analysis using PCR, microarray or sequencing.
Materials and Reagents
- 500 ml of log-phase yeast cell culture per ChIP (at -0.9 x 107 cells/ml, ~4.5 x 109 cells per sample)
- 37% formaldehyde
- 2.5 M glycine in H2O (heat sterilized)
- Liquid nitrogen, dry ice/ethanol bath or -70 °C freezer
- 0.5 mm Zirconia/Silica Beads (Bio Spec Products, catalog number: 11079105z )
- Commercial protease inhibitor cocktails, for example:
Roche Complete protease inhibitor cocktail tablets (F. Hoffmann-La Roche, catalog number: 11697498001 )
Roche Complete Mini protease inhibitor cocktail tablets (F. Hoffmann-La Roche, catalog number: 11836153001 ) - EZview anti-Myc affinity gel (red colored beads) (Sigma-Aldrich, catalog number: E6654 )
- Minelute kit for final DNA purification (QIAGEN, catalog number: 28004 )
- LiCl
- NaOAc
- Ethanol
- Triton X-100
- TE
- NaCl
- EDTA
- Isopropanol
- NP-40
- Na-deoxycholate
- SDS
- Tris-buffered saline (TBS) (10x stock) (see Recipes)
- Lysis/IP buffer (see Recipes)
- Lysis buffer/500 mM NaCl (see Recipes)
- IP wash solution (see Recipes)
- TE/1% SDS (100 ml) (see Recipes)
- TE/0.67% SDS (100 ml) (see Recipes)
- TE (100 ml) (PH 8.0) (see Recipes)
- 1 mM PMSF (Fluka, catalog number: 93482 ) (see Recipes)
Equipment
- Millipore stericup sterile vacuum filter units, 500 ml funnel, 0.22 μm or 0.45 μm pore size (EMD Millipore, catalog number: SCGVU05RE , SCHVU11RE )
- Syringe needle (BD Biosciences, catalog number: 305155 or 305156 )
- 5 ml snap-cap tubes, polypropylene (preferred) or polystyrene (BD Biosciences, Falcon®)
- 15 and 50 ml conical polypropylene screw-top tubes (BD Biosciences, Falcon®)
- Branson Sonifier 250 with microtip or Digital Sonifier S-450D (BD Biosciences)
- Refrigerated tabletop centrifuges, e.g. Beckman GS-6R, GS-15R (Beckman Coulter), or Eppendorf refrigerated multipurpose centrifuges (EppendorfTM, model: 5810R and 5804R ) (or simply put an ordinary tabletop centrifuge in the cold room)
- Fume hood
- FastPrep machine (FastPrep, catalog number: 6004500 )
- Hemacytometer
- Spectrophotometer
Procedure
Category
Molecular Biology > DNA > DNA-protein interaction
Microbiology > Microbial genetics > DNA
Biochemistry > Protein > Immunodetection
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Post a Question
