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"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.
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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 |
| Antisera (affinity-purified) | View our custom antisera service |
| 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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