Chromatin Immunoprecipitation (ChIP) Assay for Detecting Direct and Indirect Protein – DNA Interactions in Magnaporthe oryzae
利用染色质免疫共沉淀（ChIP）技术检测稻瘟病菌中蛋白质-DNA的直接与间接相互作用
发布: 2015年11月05日第5卷第21期 DOI: 10.21769/BioProtoc.1643 浏览次数: 14876
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参见作者原研究论文
The authors used this protocol in:
Oct 2014
Abstract
Chromatin immunoprecipitation (ChIP) is a powerful technology for analyzing protein-DNA interactions in cells. Robust ChIP procedures have been established for investigating direct interactions between protein and DNA. However, detecting indirect protein-DNA interactions in vivo is challenging. Recently, we used ChIP to analyze an indirect protein-DNA interaction between a putative histone demethylase, MoJmjC, and the promoter of the superoxide dismutase 1-encoding gene MoSOD1 in the rice blast fungus Magnaporthe oryzae (M. oryzae) (Fernandez et al., 2014). We tagged MoJmjC with the 3x FLAG epitope (Fernandez et al., 2014), instead of the larger and more commonly used GFP epitope, to mitigate against steric hindrance. We also employed a two-step cross-linking strategy using DSG and formaldehyde-rather than the one-step formaldehyde cross-linking procedure more frequently employed for analyzing direct protein-DNA interactions - in order to better capture the indirect MoJmjC-MoSOD1 DNA interactions in vivo. In addition, we have shown that two-step cross-linking is suitable for ChIP analysis of direct protein-DNA interactions between a GATA transcription factor, Asd4, and its cognate binding site (Marroquin-Guzman and Wilson, 2015). Here, we provide a detailed protocol for chromatin immunoprecipitation, with versatile two-step cross-linking, in M. oryzae.
Materials and Reagents
- Oak Ridge Centrifuge Tubes (Thermo Fisher Scientific, NalgenetmTM, catalog number: 3118-0050 )
- Miracloth (EMD Millipore Corporation, catalog number: 475855-1R )
- Saccharomyces cerevisiae strain XK1-25: MATa trp1 (kindly provided by Dr. Jin-Rong Xu, Dept. of Botany and Plant Pathology, Purdue University)
- pHZ126 yeast shuttle vector (kindly provided by Dr. Jin-Rong Xu, Dept. of Botany and Plant Pathology, Purdue University, USA)
- QIAquick gel extraction kit (QIAGEN, catalog number: 28704 )
- Alkali-cation yeast transformation kit (MP Biomedicals, catalog number: 2200-200 )
- QIAprep Spin Miniprep Kit (QIAGEN, catalog number: 27104 )
- UltraClean Midi Prep Kit (MO BIO Laboratories, catalog number: 12700-20 )
- Zymoprep Yeast Plasmid Mini Prep II Kit (Epigenetics, catalog number: D2004 )
- Formaldehyde (Thermo Fisher Scientific, PierceTM, catalog number: 28906 )
- DSG (Thermo Fisher Scientific, catalog number: 20593 )
- CelLyticTM PN Plant Nuclear Isolation Kit (Sigma-Aldrich, catalog number: CELLYTPN1 )
- Nuclei isolation buffer (NIB) (included in the CelLyticTM PN Plant Nuclear Isolation Kit)
- Triton X-100 Solution (10% in H2O) (MBL International, catalog number: JM-2104-100 )
- Proteinase inhibitor cocktail (Sigma-Aldrich, catalog number: P8215 )
- Deoxycholate (Sigma-Aldrich, catalog number: D6750 )
- Sodium butyrate (Sigma-Aldrich, catalog number: B5887 )
- Sepharose 4B beads (Sigma-Aldrich, catalog number: CL4B200 )
- ANTI-FLAG® M2 Affinity Gel (Sigma-Aldrich, catalog number: A2220 )
- Anti-IgG agarose (Sigma-Aldrich, catalog number: A0919 )
- 3x FLAG® Peptide (Sigma-Aldrich, catalog number: F4799 )
- Glycine (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15527-013 )
- Proteinase K (Thermo Fisher Scientific, catalog number: EO0491 )
- RNase A (Thermo Fisher Scientific, catalog number: EN0531 )
- Wizard PCR Clean-up kit (Promega Corporation, catalog number: A9281 )
- Yeast nitrogen base without amino acids (Difco, catalog number: 0919-15 )
- Trp DO supplement (Clontech, catalog number: 630413 )
- Disuccinimidyl glutarate (DSG) (Thermo Fisher Scientific, catalog number: 20593 )
- Lysing Enzymes from Trichoderma harzianum (Sigma-Aldrich, catalog number: L1412 )
- Hygromycin B (Thermo Fisher Scientific, GibcoTM, catalog number: 10-687-010 )
- SD-Trp solid medium (see Recipes)
- SD-Trp liquid solution (see Recipes)
- LB agar plates supplemented with 100 μg/ml ampicillin (see Recipes)
- Liquid complete medium (see Recipes)
- DSG cross-linking buffer (see Recipes)
- 1x NIB (see Recipes)
- NIBA (see Recipes)
- Nuclear lysis buffer (see Recipes)
- HEPES-buffered saline (HBS) (see Recipes)
- 0.1 M glycine HCl (pH 3.5) (see Recipes)
- Elution buffer (see Recipes)
- OM buffer (see Recipes)
- ST buffer (see Recipes)
- STC buffer (see Recipes)
- PTC buffer (see Recipes)
- Bottom medium (see Recipes)
- Top medium (see Recipes)
Equipment
- Benchtop shakers (Eppendorf, catalog number: M1354-0000 )
- VirTis VirSonic 50 Ultrasonic Cell Disrupter Disruptor (VirTis company)
- Centrifuge (Eppendorf, model: 5415D )
- Mini-Tube Rotators (Thermo Fisher Scientific, Fisher Scientific, catalog number: 05-450-127 )
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Li, G., Marroquin-Guzman, M. and Wilson, R. A. (2015). Chromatin Immunoprecipitation (ChIP) Assay for Detecting Direct and Indirect Protein – DNA Interactions in Magnaporthe oryzae. Bio-protocol 5(21): e1643. DOI: 10.21769/BioProtoc.1643.
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分类
微生物学 > 微生物生物化学 > 蛋白质
生物化学 > 蛋白质 > 免疫检测
分子生物学 > DNA > DNA-蛋白质相互作用
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