17-245 Acetyl-Histone H3 Immunoprecipitation (ChIP) Assay Kit

17-245
1 kit  Kit capacity: 22 assays
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      Description
      Catalogue Number17-245
      Brand Family Upstate
      Trade Name
      • Upstate
      DescriptionAcetyl-Histone H3 Immunoprecipitation (ChIP) Assay Kit
      OverviewFor use to immunoprecipitate transcriptionally active chromatin from mammalian cells using anti-acetyl-Histone H3. Detection of the gene or promoter of interest in immunoprecipitated chromatin must be empirically determined by the researcher using quantitative PCR or Southern slot-blot analysis with promotor specific primers or probe.
      References
      Product Information
      Components
      • Anti-acetyl-Histone H3 (Cat.# 06-599)
      • Protein A agarose/Salmon Sperm DNA (Cat.# 16-157)
      • All necessary buffers.
      Quality LevelMQ100
      Applications
      ApplicationFor use to immunoprecipitate transcriptionally active chromatin from mammalian cells using anti-acetyl-Histone H3.
      Key Applications
      • Immunoprecipitation
      Biological Information
      Entrez Gene Number
      Entrez Gene SummaryHistones are basic nuclear proteins that are responsible for the nucleosome structure of the chromosomal fiber in eukaryotes. Nucleosomes consist of approximately 146 bp of DNA wrapped around a histone octamer composed of pairs of each of the four core histones (H2A, H2B, H3, and H4). The chromatin fiber is further compacted through the interaction of a linker histone, H1, with the DNA between the nucleosomes to form higher order chromatin structures. This gene is intronless and encodes a member of the histone H3 family. Transcripts from this gene lack polyA tails; instead, they contain a palindromic termination element. This gene is located separately from the other H3 genes that are in the histone gene cluster on chromosome 6p22-p21.3.
      Gene Symbol
      • HIST3H3
      • H3FT
      • H3/t
      • H3t
      • MGC126888
      • H3/g
      • H3.4
      • H3T
      • MGC126886
      Modifications
      • Acetylation
      UniProt Number
      UniProt SummaryFUNCTION: SwissProt: Q16695 # Core component of nucleosome. Nucleosomes wrap and compact DNA into chromatin, limiting DNA accessibility to the cellular machineries which require DNA as a template. Histones thereby play a central role in transcription regulation, DNA repair, DNA replication and chromosomal stability. DNA accessibility is regulated via a complex set of post-translational modifications of histones, also called histone code, and nucleosome remodeling.
      SIZE: 136 amino acids; 15508 Da
      SUBUNIT: The nucleosome is a histone octamer containing two molecules each of H2A, H2B, H3 and H4 assembled in one H3-H4 heterotetramer and two H2A-H2B heterodimers. The octamer wraps approximately 147 bp of DNA.
      SUBCELLULAR LOCATION: Nucleus.
      TISSUE SPECIFICITY: Expressed in testicular cells.
      DEVELOPMENTAL STAGE: Expressed during S phase, then expression strongly decreases as cell division slows down during the process of differentiation.
      PTM: Acetylation is generally linked to gene activation. Acetylation on Lys-10 impairs methylation at Arg-9. Acetylation on Lys-19 and Lys-24 favors methylation at Arg-18 (By similarity). & Citrullination at Arg-9 and/or Arg-18 by PADI4 impairs methylation and represses transcription (By similarity). & Asymmetric dimethylation at Arg-18 by CARM1 is linked to gene activation. Symmetric dimethylation at Arg-9 by PRMT5 is linked to gene repression (By similarity). & Methylation at Lys-5, Lys-37 and Lys-80 are linked to gene activation. Methylation at Lys-5 facilitates subsequent acetylation of H3 and H4. Methylation at Lys-80 is associated with DNA double-strand break (DSB) responses and is a specific target for TP53BP1. Methylation at Lys-10 and Lys-28 are linked to gene repression. Methylation at Lys-10 is a specific target for HP1 proteins (CBX1, CBX3 and CBX5) and prevents subsequent phosphorylation at Ser-11 and acetylation of H3 and H4. Methylation at Lys-5 and Lys-80 require preliminary monoubiquitination of H2B at 'Lys-120'. Methylation at Lys-10 and Lys-28 are enriched in inactive X chromosome chromatin (By similarity). & Phosphorylated at Thr-4 by GSG2/haspin during prophase and dephosphorylated during anaphase. At centromeres, specifically phosphorylated at Thr-12 from prophase to early anaphase. Phosphorylated at Ser-11 during the whole mitosis. Phosphorylation at Ser-11, which is linked to gene activation, prevents methylation at Lys-10 but facilitates acetylation of H3 and H4. Phosphorylated at Ser-29 by MLTK isoform 1, RPS6KA5 or AURKB during mitosis or upon ultraviolet B irradiation (By similarity). & Phosphorylation at 'Ser-11' is crucial for chromosome condensation and cell-cycle progression during mitosis and meiosis. In addition phosphorylation at 'Ser-11' is important during interphase because it enables the transcription of genes following external stimulation, like stress or growth factors. Phosphorylation at 'Ser-11' is also an essential regulatory mechanism for neoplastic cell transformation. Phosphorylation at 'Ser-11' by AURKB/Aurora-B mediates the dissociation of HP1 proteins (CBX1, CBX3 and CBX5) from heterochromatin. & Ubiquitinated (By similarity).
      SIMILARITY: SwissProt: Q16695 ## Belongs to the histone H3 family.
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Usage Statement
      • Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
      Storage and Shipping Information
      Packaging Information
      Material Size1 kit
      Material PackageKit capacity: 22 assays
      Transport Information
      Supplemental Information
      Specifications

      Documentation

      Acetyl-Histone H3 Immunoprecipitation (ChIP) Assay Kit SDS

      Title

      Safety Data Sheet (SDS) 

      Acetyl-Histone H3 Immunoprecipitation (ChIP) Assay Kit Certificates of Analysis

      TitleLot Number
      Acetyl-Histone H3 Immunoprecipitation (ChIP)2475683
      Acetyl-Histone H3 Immunoprecipitation (ChIP) Assay Kit3043203
      Acetyl-Histone H3 Immunoprecipitation (ChIP) Assay Kit3125658
      Acetyl-Histone H3 Immunoprecipitation (ChIP) Assay Kit2989600
      Acetyl-Histone H3 Immunoprecipitation (ChIP) Assay Kit2862343
      Acetyl-Histone H3 Immunoprecipitation (ChIP) Assay Kit2932691
      Acetyl-Histone H3 Immunoprecipitation (ChIP) Assay Kit - 23904932390493
      Acetyl-Histone H3 Immunoprecipitation (ChIP) Assay Kit - 23957122395712
      Acetyl-Histone H3 Immunoprecipitation (ChIP) Assay Kit - 06100429040610042904
      Acetyl-Histone H3 Immunoprecipitation (ChIP) Assay Kit - 1777717777

      References

      Reference overviewApplicationSpeciesPub Med ID
      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)Human18628788 18628788
      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 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 17213810
      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 17855562
      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 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 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 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 15039422
      p53-dependent inhibition of FKHRL1 in response to DNA damage through protein kinase SGK1.
      Han You, YingJu Jang, Annick Itie You-Ten, Hitoshi Okada, Jennifer Liepa, Andrew Wakeham, Kathrin Zaugg, Tak W Mak
      Proceedings of the National Academy of Sciences of the United States of America  101  14057-62  2004

      Show Abstract Full Text Article
      15383658 15383658
      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 15087456

      FAQ

      QuestionAnswer
      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.
      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.
      Why do you use Salmon Sperm DNA to block the agarose? Won't my PCR react with the Salmon Sperm DNA in my sample?Salmon Sperm is used to reduce the non-specific interaction of chromatin DNA with the agarose. It is unlikely that people will be performing ChIP from salmon tissues, so the DNA shouldn't amplify with your PCR primers due to cross-hybridization.
      Which promoter do I include on my primer to look for gene activation?GAPDH is a good control if you are using an antibody specific for acetylated Histones.

      Related Products & Applications

      Kit Component

      Catalog Number Description  
      16-157 Protein A Agarose/Salmon Sperm DNA, 2.5 mL Show Pricing & Availability

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      Life Science Research > Protein Detection and Quantification > Immunoassays > Immunoprecipitation (IP) > Chromatin Immunoprecipitation (ChIP) > ChIP Kits & Reagents
      Life Science Research > Antibodies and Assays > Immunoassays > Immunoprecipitation (IP) > Chromatin Immunoprecipitation (ChIP) > ChIP Kits & Reagents