17-245 Sigma-AldrichAcetyl-Histone H3 Immunoprecipitation (ChIP) Assay Kit
For use to immunoprecipitate transcriptionally active chromatin from mammalian cells using anti-acetyl-Histone H3.
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Overview
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Quality Level | MQ100 |
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Application | For use to immunoprecipitate transcriptionally active chromatin from mammalian cells using anti-acetyl-Histone H3. |
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Material Size | 1 kit |
Material Package | Kit capacity: 22 assays |
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Catalog Number | GTIN |
17-245 | 04053252017155 |
Documentation
Acetyl-Histone H3 Immunoprecipitation (ChIP) Assay Kit SDS
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Acetyl-Histone H3 Immunoprecipitation (ChIP) Assay Kit Certificates of Analysis
References
Reference overview | Application | Species | Pub Med ID |
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Epigenetic analysis reveals a euchromatic configuration in the FMR1 unmethylated full mutations. Tabolacci, E; Moscato, U; Zalfa, F; Bagni, C; Chiurazzi, P; Neri, G European journal of human genetics : EJHG 16 1487-98 2008 Show Abstract | Chromatin Immunoprecipitation (ChIP) | Human | 18628788 |
Inhibition of histone deacetylase activity induces developmental plasticity in oligodendrocyte precursor cells. Costas A Lyssiotis,John Walker,Chunlei Wu,Toru Kondo,Peter G Schultz,Xu Wu Proceedings of the National Academy of Sciences of the United States of America 104 2007 Show Abstract Full Text Article | 17855562 | ||
Chromatin remodeling at the Th2 cytokine gene loci in human type 2 helper T cells. Takaaki Kaneko,Hiroyuki Hosokawa,Masakatsu Yamashita,Chrong-Reen Wang,Akihiro Hasegawa,Motoko Y Kimura,Masayuki Kitajiama,Fumio Kimura,Masaru Miyazaki,Toshinori Nakayama Molecular immunology 44 2007 Show Abstract | 17166591 | ||
Epigenetic patterns of the retinoic acid receptor beta2 promoter in retinoic acid-resistant thyroid cancer cells. Cras, A, et al. Oncogene, 26: 4018-24 (2007) 2007 Show Abstract | 17213810 | ||
Critical YxKxHxxxRP motif in the C-terminal region of GATA3 for its DNA binding and function. Ryo Shinnakasu, Masakatsu Yamashita, Kenta Shinoda, Yusuke Endo, Hiroyuki Hosokawa, Akihiro Hasegawa, Shinji Ikemizu, Toshinori Nakayama Journal of immunology (Baltimore, Md. : 1950) 177 5801-10 2006 Show Abstract | 17056504 | ||
Ras-ERK MAPK cascade regulates GATA3 stability and Th2 differentiation through ubiquitin-proteasome pathway. Masakatsu Yamashita, Ryo Shinnakasu, Hikari Asou, Motoko Kimura, Akihiro Hasegawa, Kahoko Hashimoto, Naoya Hatano, Masato Ogata, Toshinori Nakayama The Journal of biological chemistry 280 29409-19 2005 Show Abstract | 15975924 | ||
Differential expression of IFN regulatory factor 4 gene in human monocyte-derived dendritic cells and macrophages. Anne Lehtonen, Ville Veckman, Tuomas Nikula, Riitta Lahesmaa, Leena Kinnunen, Sampsa Matikainen, Ilkka Julkunen Journal of immunology (Baltimore, Md. : 1950) 175 6570-9 2005 Show Abstract | 16272311 | ||
CD28 costimulation controls histone hyperacetylation of the interleukin 5 gene locus in developing th2 cells. Masamichi Inami, Masakatsu Yamashita, Yoshiyuki Tenda, Akihiro Hasegawa, Motoko Kimura, Kahoko Hashimoto, Nobuo Seki, Masaru Taniguchi, Toshinori Nakayama The Journal of biological chemistry 279 23123-33 2004 Show Abstract | 15039422 | ||
Essential role of GATA3 for the maintenance of type 2 helper T (Th2) cytokine production and chromatin remodeling at the Th2 cytokine gene loci. Masakatsu Yamashita, Maki Ukai-Tadenuma, Takeshi Miyamoto, Kaoru Sugaya, Hiroyuki Hosokawa, Akihiro Hasegawa, Motoko Kimura, Masaru Taniguchi, James DeGregori, Toshinori Nakayama The Journal of biological chemistry 279 26983-90 2004 Show Abstract | 15087456 | ||
Cyclin D1 activation in B-cell malignancy: association with changes in histone acetylation, DNA methylation, and RNA polymerase II binding to both promoter and distal sequences. Hui Liu, Jin Wang, Elliot M Epner Blood 104 2505-13 2004 Show Abstract | 15226187 |
FAQ
Question | Answer |
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How should I resuspend my pellet prior to PCR? | You should resuspend your pellet in water and not TE as the EDTA found in the TE may interfere with PCR. |
Is there ever a time when I do not need to cross-link Histones? | In native ChIP, Histone H3 and Histone H4 do not need to be crosslinked as they are very tightly associated. Histone H2A and Histone H2B are not as tightly associated, but will still work in native ChIP. |
What were your conditions for PCR? | Please see the manual for The EZ ChIP Kit (Catalog #17-371) for more information. |
If I wanted to quantitate my immunoprecipitated DNA, how would I do so? | DNA purified from ChIP experiments can be quantitated by PCR, providing the amplifying oligos meet specific criteria. Oligos should be 24 mers, with a GC content of 50% (+/- 4) and a Tm of 60.0C (+/- 2.0). You must be certain that the PCR reactions are within the linear range of amplification. Generally it takes time to achieve this. Too much input DNA will affect your results, so set up several tubes for each experiment to optimize the input DNA. Generally, this is about 1/25th to 1/100th for yeast, approximately 1/10 for mammalian cells, but depends on the amount of antibody and input chromatin. Also, do not use more than 20 cycles, making sure that dNTP's always remain in excess. Also, include each reaction a control primer (to compare your experimental band against-make sure the sizes are sufficiently different to allow proper separation-75 base pairs is usually OK) set to a region of the genome that should not change throughout your experimental conditions. Also PCR from purified input DNA (no ChIP) and include no antibody control PCR's as well. PCR products should be no more than 500 base pairs and should span the area of interest (where you think you will see changes in acetylation or methylation of histones). All PCR products should be run on 7-8% acrylamide gels and stained with SYBR Green 1 (Molecular Probes) at a dilution of 1:10,000 (in 1X Tris-borate-EDTA buffer, pH 7.5) for 30 minutes-no destaining is required. Quantitation is carried out subsequent to scanning of the gel on a Molecular Dynamics Storm 840 or 860 in Blue fluorescence mode with PMT voltage at 900 with ImageQuant software. This has distinct advantages over ethidium bromide staining. SYBR Green is much more sensitive, and illumination of ethidium stained gels can vary across the gel based on the quality of UV bulbs in your in your light box. For further info, see Strahl-Bolsinger et al. (1997) Genes Dev. 11: 83-93. A radioactive quantitation m |
I am not getting amplification with input DNA. What did I do wrong? | Your input DNA sample should be taken just prior to adding the antibody. It is considered the starting material. If you are not seeing amplification with your input DNA, either you have not successfully reversed the cross links or the PCR is not working for reasons other than the kit. |
How would you recommend eluting Antibody-protein-DNA complexes from agarose (or sepharose) in order to perform a Re-ChIP experiment? | The complex is removed with the elution buffer that you find in the ChIP assay kit. For a re-CHIP, it might make sense to add protease inhibitors to the IP wash buffers and the elution buffer and the second set of dilution buffers. Make sure everything stays cold so that the proteins aren't degraded during the collection of the first complex or during the second IP. |
Do you have any tips for sonication? | Keep cells on ice throughout the procedure - even during sonication. Be sure that you don't sonicate for to long (more than 30 seconds could cause sample overheating and denaturation). |
Why is more DNA is precipitated in my no-antibody control than for my test sample? | To eliminate banding in your negative controls you can do several things: A) Pre-clear the 2ml diluted cell pellet suspension with 80 microliters of Salmon Sperm DNA/Protein A Agarose-50% Slurry for 30 minutes at 4ºC with agitation. You could try to preclear the lysate longer or with more clearings. B) Titrate your input DNA, to see when the bands in the NFA disappear. C) Use an alternative lysis procedure: Resuspend cell pellet in 200 microliters of 5mM Pipes pH 8.0, 85mM KCl, 0.5% NP40 containing protease inhibitors. Place on ice for 10 minutes. Pellet by centrifugation (5 minutes at 5000 rpm). Resuspend pellet in 200 microliters of 1% SDS, 10mM EDTA, 50mM Tris-HCl, pH 8.1 containing protease inhibitors. Incubate on ice for 10 minutes. D) Block the Salmon Sperm DNA Agarose prior to use in 1-5% BSA and Chip dilution buffer (mix at room temperature for 30 minutes). After incubation, spin the agarose and remove the 1% BSA/ChIP assay buffer supernatant. Wash once in ChIP assay buffer and continue. |
What is 'Input DNA', and why no 'Output DNA'? | Input DNA is DNA obtained from chromatin that has been cross-link reversed similar to your samples. It is a control for PCR effectiveness. Output DNA is the DNA from each of your ChIP experiments. |
What types of controls do I need to run in the IP and the PCR portions of the ChIP? | ChIP control: use Anti-acetyl H3 primary antibody and PCR for the GAPDH gene promoter. This will ensure that each step of the procedure is working. PCR amplification: Control for PCR amplification using primers designed against a sequence that would not be enriched by your chromatin IP. Liner Range PCR controls: Ensure that PCR amplification is in the linear range by setting up each reaction at different dilutions of DNA for various amplification cycle numbers, and select the final PCR conditions accordingly. The assays are typically done in duplicate or triplicate. The average fragment size after sonication is ~500 bp (Kondo, et al. Molecular and Cellular Biology, January 2003, p. 206-215, Vol. 23, No. 1) Treatment controls: 1) ChIP analysis of a transcribed region of the gene of interest which is >40 kb away from the promoter you are looking at. This may reveal that the activation level (e.g., acetylation level) may be very low or more importantly, not affected by your treatment. 2) Control for specificity of an induced local Histone hyperacetylation, you could analyze the acetylation level of another promoter (Sachs, et al. Proc. Natl. Acad. Sci. USA 97:2000, 13138-13143). No primary antibody control: This is the control in which you run the ChIP assay but don't add the primary immunoprecipitating antibody. It will ensure that you are not seeing sequences that bind non-specifically to the beads and that the recognition of your protein by the antibody you are using is required for enrichment of the target sequence Negative antibody control: A normal serum, normal IgG, or an antibody to a distant protein (all from the same species) is a good negative antibody control. The best control if using a polyclonal antibody is pre-immune antiserum of the animal that has been immunized. |