17-229 Acetyl-Histone H4 Immunoprecipitation (ChIP) Assay Kit

17-229
1 kit  Kit capacity: 22 assays
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      Description
      Catalogue Number17-229
      Brand Family Upstate
      Trade Name
      • Upstate
      DescriptionAcetyl-Histone H4 Immunoprecipitation (ChIP) Assay Kit
      OverviewFor use to immunoprecipitate transcriptionally active chromatin from mammalian cells using anti-Acetyl-Histone H4, ChIP grade rabbit antiserum. 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, using promotor specific primers or probe.
      References
      Product Information
      Components
      • Anti-acetyl-Histone H4 (Cat.# 06-866)
      • Protein A agarose/Salmon Sperm DNA (Cat.# 16-157)
      • All necessary buffers
      Applications
      ApplicationAcetyl-Histone H4 Immunoprecipitation (ChIP) Assay Kit used to immunoprecipitate transcriptionally active chromatin from mammalian cells using anti-Acetyl-Histone H4, ChIP grade rabbit antiserum.
      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. Two molecules of each of the four core histones (H2A, H2B, H3, and H4) form an octamer, around which approximately 146 bp of DNA is wrapped in repeating units, called nucleosomes. The linker histone, H1, interacts with linker DNA between nucleosomes and functions in the compaction of chromatin into higher order structures. This gene is intronless and encodes a member of the histone H4 family. Transcripts from this gene lack polyA tails but instead contain a palindromic termination element. This gene is found in the large histone gene cluster on chromosome 6.
      Gene Symbol
      • HIST1H4A
      • H4/J
      • HIST1H4F
      • HIST1H4L
      • H4FN
      • H4FA
      • H4FH
      • HIST1H4H
      • H4F2
      • H4FM
      • H4/A
      • H4FK
      • H4FG
      • HIST2H4
      • H4FB
      • H4/K
      • HIST1H4I
      • H4/N
      • HIST1H4B
      • H4FE
      • H4/M
      • H4/a
      • HIST1H4E
      • HIST4H4
      • HIST2H4A
      • H4FC
      • H4FI
      • H4/H
      • HIST1H4C
      • H4/G
      • HIST1H4K
      • H4FJ
      • H4FD
      • H4/I
      • H4/B
      • H4/D
      • H4/C
      • HIST1H4J
      • HIST1H4D
      • H4/E
      Modifications
      • Acetylation
      UniProt Number
      UniProt SummaryFUNCTION: SwissProt: P62805 # 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: 103 amino acids; 11367 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.
      PTM: Symmetric dimethylation on Arg-4 by the PRDM1/PRMT5 complex may play a crucial role in the germ-cell lineage (By similarity). & Ubiquitinated by the CUL4-DDB-RBX1 complex in response to ultraviolet irradiation. This may weaken the interaction between histones and DNA and facilitate DNA accessibility to repair proteins.
      SIMILARITY: SwissProt: P62805 ## Belongs to the histone H4 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 H4 Immunoprecipitation (ChIP) Assay Kit MSDS

      타이틀

      물질안전보건자료(MSDS) 

      Acetyl-Histone H4 Immunoprecipitation (ChIP) Assay Kit Certificates of Analysis

      TitleLot Number
      Acetyl-Histone H4 Immunoprecipitation (ChIP)2475819
      Acetyl-Histone H4 Immunoprecipitation (ChIP) Assay Kit2863908
      Acetyl-Histone H4 Immunoprecipitation (ChIP) Assay Kit3043204
      Acetyl-Histone H4 Immunoprecipitation (ChIP) Assay Kit3099101
      Acetyl-Histone H4 Immunoprecipitation (ChIP) Assay Kit - 21351612135161
      Acetyl-Histone H4 Immunoprecipitation (ChIP) Assay Kit - 06110459430611045943
      Acetyl-Histone H4 Immunoprecipitation (ChIP) Assay Kit - 1704317043
      Acetyl-Histone H4 Immunoprecipitation (ChIP) Assay Kit - 1810918109
      Acetyl-Histone H4 Immunoprecipitation (ChIP) Assay Kit - 1902319023
      Acetyl-Histone H4 Immunoprecipitation (ChIP) Assay Kit - 1933719337

      References

      Reference overviewApplicationSpeciesPub Med ID
      The inhibitor of histone deacetylases sodium butyrate enhances the cytotoxicity of mitomycin C.
      Anastas Gospodinov,Stanislava Popova,Ivelina Vassileva,Boyka Anachkova
      Molecular cancer therapeutics  11  2012

      요약 표시
      22891039 22891039
      SUV420H2-mediated H4K20 trimethylation enforces RNA polymerase II promoter-proximal pausing by blocking hMOF-dependent H4K16 acetylation.
      Kapoor-Vazirani, P; Kagey, JD; Vertino, PM
      Molecular and cellular biology  31  1594-609  2011

      요약 표시 기사 전문
      21321083 21321083
      A possible inflammatory role of twist1 in human white adipocytes.
      Pettersson AT, Laurencikiene J, Mejhert N, Näslund E, Bouloumié A, Dahlman I, Arner P, Rydén M
      Diabetes  59  564-71. Epub 2009 Dec 10.  2010

      요약 표시 기사 전문
      20007935 20007935
      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

      요약 표시
      Chromatin Immunoprecipitation (ChIP)Human18628788 18628788
      Repressor element-1 silencing transcription factor/neuronal restrictive silencer factor (REST/NRSF) can regulate HSV-1 immediate-early transcription via histone modification.
      Pinnoji, RC; Bedadala, GR; George, B; Holland, TC; Hill, JM; Hsia, SC
      Virology journal  4  56  2007

      요약 표시 기사 전문
      17555596 17555596
      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

      요약 표시
      15226187 15226187
      Counterregulation of chromatin deacetylation and histone deacetylase occupancy at the integrated promoter of human immunodeficiency virus type 1 (HIV-1) by the HIV-1 repressor YY1 and HIV-1 activator Tat
      He, G. and Margolis, D. M.
      Mol Cell Biol, 22:2965-73 (2002)  2002

      Chromatin Immunoprecipitation (ChIP)11940654 11940654

      Brochure

      Title
      Shaping Epigenetics Discovery - Epigenetics Product Selection Brochure

      FAQ

      QuestionAnswer
      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.

      관련 제품 및 어플리케이션

      Kit Component

      카탈로그 번호 설명  
      06-866 Anti-acetyl-Histone H4 Antibody 가격 및 재고 조회
      16-157 Protein A Agarose/Salmon Sperm DNA, 2.5 mL 가격 및 재고 조회

      제품 패밀리

      카테고리

      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