Molecular Biology

ChIP

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View the protocol "Chromatin Immunoprecipitation (ChIP) on Unfixed Chromatin from Cells and Tissues to Analyze Histone Modifications" at BioSupplynet.com

Alexandre Wagschal, Katia Delaval, Maëlle Pannetier, Philippe Arnaud, and Robert Feil—Institute of Molecular Genetics, CNRS and University of Montpellier, 34293 Montpellier, France

ABSTRACT
In cells and tissues, the histone proteins that constitute the nucleosomes can present multiple post-translational modifications, such as lysine acetylation, lysine and arginine methylation, serine phosphorylation, and lysine ubiquitination. On their own, or in combination, these covalent modifications on the core histones are thought to play essential roles in chromatin organization and gene expression in eukaryotes. Importantly, patterns of histone modifications may be somatically conserved and can, thereby, maintain locus-specific repression/activity in defined lineages, or throughout development. Indirect immunofluorescence studies on cultured cells have been pivotal in unraveling the roles of histone modifications. However, to address in detail what happens at specific sites in vivo, chromatin immunoprecipitation (ChIP) is the method of choice. Here, we describe how ChIP can be performed on non-fixed chromatin from animal cells or tissues (fresh or frozen) to analyze histone modifications at specific chromosomal sites. These protocols are suitable only for analyzing histones and their modifications. For other applications, chromatin immunoprecipitation should be performed on cross-linked chromatin.

Products Available for this Protocol

Protocol Material Description Product #  Product Name Add to Cart
100-bp DNA step ladder D5042 123 bp Ladder, buffered aqueous solution
Agarose A9539 Agarose, For routine use
Xylene cyanol FF X4126 Xylene Cyanol FF for molecular biology
Bromophenol blue B5525 Bromophenol Blue sodium salt for electrophoresis
Ethidium bromide solution E1385 Ethidium bromide solution, for molecular biology, 500 µg/mL in H2O, suitable for gel electrophoresis and DNA isolation procedures
Cell culture medium View our entire line of media and salts
TBE buffer T4073 Tris-Borate-EDTA buffer, working solution, Biotechnology Performance Certified
Dithiothreitol (DTT) D9779 DL-Dithiothreitol, for molecular biology, ≥98% (TLC),
≥99% (titration)

Tris-Cl 93313 Trizma® hydrochloride buffer solution, BioChemika Ultra, for molecular biology, pH 7.4
SDS L4390 Sodium dodecyl sulfate, for molecular biology, ≥98.5% (GC)
KCl P9541 Potassium chloride, for molecular biology, ≥99%
IGEPAL CA-630 I8896 Igepal® CA-630, for molecular biology
Phosphate-buffered saline (PBS) P5368 Phosphate buffered saline, pH 7.4, Biotechnology Performance Certified
EDTA E5134 Ethylenediaminetetraacetic acid disodium salt dihydrate, for molecular biology, ≥99%
Micrococcal nuclease N3755 Nuclease micrococcal from Staphylococcus aureus, 100-300 units/mg protein
Trypsin T6567 Trypsin from porcine pancreas, Proteomics Grade, Dimethylated
Sucrose S0389 Sucrose, for molecular biology, ≥99.5% (GC)
Aprotinin A1153 Aprotinin from bovine lung, lyophilized powder, 3-8 TIU/mg solid
Phenol:chloroform:isoamyl alcohol 25:24:1 P2069 Phenol:Chloroform:Isoamyl Alcohol 25:24:1 Saturated with 10 mM Tris, pH 8.0, 1 mM EDTA
Proteinase K P2308 Proteinase K from Tritirachium album , BioUltra, for molecular biology, >30 units/mg protein, lyophilized powder
Sodium butyrate B5887 Sodium butyrate, ~99% (GC)
Sodium bicarbonate S7277 Sodium bicarbonate, for molecular biology, 99.7-100.3%
Tris-HCl T5941 Trizma® hydrochloride, for molecular biology, Biotechnology Performance Certified, cell culture tested, ≥99.0% (titration)
Glycerol G5516 Glycerol, for molecular biology, ≥99%
Isopropanol I9516 2-Propanol, for molecular biology, ≥99%
PMSF P7626 Phenylmethanesulfonyl fluoride, ≥98.5% (GC)
NaCl S3014 Sodium chloride, for molecular biology, ≥98% (titration)
MgCl2 M4880 Magnesium chloride, ~98%, insect cell culture tested
CaCl2 C2661 Calcium chloride, plant cell culture tested, anhydrous
Ethanol E7148 Ethanol, for molecular biology, 190 proof
Ethylene glycol-bis N,N,N',N'-tetra-acetic acid (EGTA) E3889 Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid, for molecular biology, ≥97%
Protein A (e.g., CL-4B Sepharose) or protein G Sepharose CL4B200 Sepharose® CL-4B, Cross-linked

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