17-295 Chromatin Immunoprecipitation (ChIP) Assay Kit

17-295
1 kit  Kit capacity: 22 Assays
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      Overview

      Replacement Information

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      For target-specific spike-in controls that make ChIP experiments more quantitative and accurate, Click on the Related Product & Applications tab above.
      Description
      Catalogue Number17-295
      Trade Name
      • EZ-ChIP
      DescriptionChromatin Immunoprecipitation (ChIP) Assay Kit
      OverviewFor use to enrich for specific regions of chromatin that are immunoprecipitated with a specific antibody for a chromatin protein. Works with all eukaryotic cell types. Detection of the gene or promoter of interest in immunoprecipitated chromatin must be empirically determined by the researcher. Quantitative PCR detection is recommended.
      Alternate Names
      • Agarose ChIP Kit
      Background InformationChromatin Immunoprecipitation (ChIP) is a powerful technique for mapping the in vivo distribution of proteins associated with chromosomal DNA. These proteins can be histone subunits and post-translational modifications or other chromatin associated proteins such as transcription factors, chromatin regulators, etc. Additionally, ChIP can be used to identify regions of the genome associated with these proteins, or conversely, to identify proteins associated with a particular region of the genome. ChIP methodology often involves protein-DNA and protein-protein cross-linking, fragmentation of the cross-linked chromatin, and subsequent immunoprecipitation of chromatin with an antibody specific to a target protein. The DNA fragments isolated in complex with the target protein can be identified by a variety of methods including PCR, DNA microarray and DNA sequencing. Standard or quantitative PCR can be performed to verify whether a particular DNA sequence (the gene or region of the genome) is associated with the protein of interest. The combination of ChIP and promoter or genomic tiling microarrays (ChIP-chip) allows genome-wide identification of DNA-binding sites for chromatin-associated proteins with precise resolution. Alternatively, high-throughput sequencing of libraries constructed from immunoprecipitated chromosomal DNA (ChIP-Seq) is a powerful alternative to ChIP-chip in mapping the protein-DNA interactions across mammalian genomes.
      References
      Product Information
      Components
      • Protein A agarose/Salmon Sperm DNA (Cat.# 16-157)
      • All necessary buffers
      PresentationContains all necessary reagents to perform 22 individual chromatin immunoprecipitation (ChIP) reactions. Supplied buffers are sufficient to generate chromatin from up to five 15 cm plates of cultured cells, each plate providing up to 10 chromatin preparations (varies with cell and assay type).
      Quality LevelMQ100
      Applications
      ApplicationContains all necessary reagents to perform 22 individual chromatin immunoprecipitation (ChIP) reactions using inexpensive protein A agarose beads.
      Biological Information
      Analytes Available
      • Protein A
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Quality AssuranceRoutinely evaluated using anti-acetyl Histone H4 polyclonal antibody (06-866) to immunoprecipitate acetylated histone H4. Subsequent immunoblot analysis was done using the same antibody.
      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

      Chromatin Immunoprecipitation (ChIP) Assay Kit SDS

      Title

      Safety Data Sheet (SDS) 

      Chromatin Immunoprecipitation (ChIP) Assay Kit Certificates of Analysis

      TitleLot Number
      Chromatin Immunoprecipitation (ChIP) Assay Kit3092746
      Chromatin Immunoprecipitation (ChIP) Assay Kit2925314
      Chromatin Immunoprecipitation (ChIP) Assay Kit2950065
      Chromatin Immunoprecipitation (ChIP) Assay Kit3068207
      Chromatin Immunoprecipitation (ChIP) Assay Kit3125651
      Chromatin Immunoprecipitation (ChIP) Assay Kit3130863
      Chromatin Immunoprecipitation (ChIP) Assay Kit2476988
      Chromatin Immunoprecipitation (ChIP) Assay Kit2472909
      Chromatin Immunoprecipitation (ChIP) Assay Kit2479785
      Chromatin Immunoprecipitation (ChIP) Assay Kit2474911

      References

      Reference overviewApplicationSpeciesPub Med ID
      HDAC1 and HDAC3 underlie dynamic H3K9 acetylation during embryonic neurogenesis and in schizophrenia-like animals.
      Večeřa, J; Bártová, E; Krejčí, J; Legartová, S; Komůrková, D; Rudá-Kučerová, J; Štark, T; Dražanová, E; Kašpárek, T; Šulcová, A; Dekker, FJ; Szymanski, W; Seiser, C; Weitzer, G; Mechoulam, R; Micale, V; Kozubek, S
      J Cell Physiol  233  530-548  2018

      Show Abstract
      28300292 28300292
      Differential in Vitro Biological Action, Coregulator Interactions, and Molecular Dynamic Analysis of Bisphenol A (BPA), BPAF, and BPS Ligand-ERα Complexes.
      Li, Y; Perera, L; Coons, LA; Burns, KA; Tyler Ramsey, J; Pelch, KE; Houtman, R; van Beuningen, R; Teng, CT; Korach, KS
      Environ Health Perspect  126  017012  2018

      Show Abstract
      29389661 29389661
      Tumor-secreted Pros1 inhibits macrophage M1 polarization to reduce antitumor immune response.
      Ubil, E; Caskey, L; Holtzhausen, A; Hunter, D; Story, C; Earp, HS
      J Clin Invest  128  2356-2369  2018

      Show Abstract
      29708510 29708510
      RGC-32 (Response Gene to Complement 32) Deficiency Protects Endothelial Cells From Inflammation and Attenuates Atherosclerosis.
      Cui, XB; Luan, JN; Dong, K; Chen, S; Wang, Y; Watford, WT; Chen, SY
      Arterioscler Thromb Vasc Biol  38  e36-e47  2018

      Show Abstract
      29449334 29449334
      Nuclear IGF-1R interacts with regulatory regions of chromatin to promote RNA polymerase II recruitment and gene expression associated with advanced tumor stage.
      Aleksic, T; Gray, NE; Wu, X; Rieunier, G; Osher, E; Mills, J; Verrill, C; Bryant, RJ; Han, C; Hutchinson, K; Lambert, A; Kumar, R; Hamdy, FC; Weyer-Czernilofsky, U; Sanderson, M; Bogenrieder, T; Taylor, S; Macaulay, VM
      Cancer Res  0  2018

      Show Abstract
      29735545 29735545
      miR-4725-3p targeting Stim1 signaling is involved in xanthohumol inhibition of glioma cell invasion.
      Ho, KH; Chang, CK; Chen, PH; Wang, YJ; Chang, WC; Chen, KC
      J Neurochem  0  2018

      Show Abstract
      29747239 29747239
      Tissue-specific CTCF-cohesin-mediated chromatin architecture delimits enhancer interactions and function in vivo.
      Hanssen, LLP; Kassouf, MT; Oudelaar, AM; Biggs, D; Preece, C; Downes, DJ; Gosden, M; Sharpe, JA; Sloane-Stanley, JA; Hughes, JR; Davies, B; Higgs, DR
      Nat Cell Biol  19  952-961  2017

      Show Abstract
      28737770 28737770
      The chromatin remodelling factor ATRX suppresses R-loops in transcribed telomeric repeats.
      Nguyen, DT; Voon, HPJ; Xella, B; Scott, C; Clynes, D; Babbs, C; Ayyub, H; Kerry, J; Sharpe, JA; Sloane-Stanley, JA; Butler, S; Fisher, CA; Gray, NE; Jenuwein, T; Higgs, DR; Gibbons, RJ
      EMBO Rep  18  914-928  2017

      Show Abstract
      28487353 28487353
      DNA methylation of intragenic CpG islands depends on their transcriptional activity during differentiation and disease.
      Jeziorska, DM; Murray, RJS; De Gobbi, M; Gaentzsch, R; Garrick, D; Ayyub, H; Chen, T; Li, E; Telenius, J; Lynch, M; Graham, B; Smith, AJH; Lund, JN; Hughes, JR; Higgs, DR; Tufarelli, C
      Proc Natl Acad Sci U S A  114  E7526-E7535  2017

      Show Abstract
      28827334 28827334
      Transcription factor NF-kappa B represses ANT1 transcription and leads to mitochondrial dysfunctions.
      Zhang, C; Jiang, H; Wang, P; Liu, H; Sun, X
      Sci Rep  7  44708  2017

      Show Abstract
      28317877 28317877

      FAQ

      QuestionAnswer
      Why is chromatin important?Any process that uses DNA as a template (replication, transcription, mitosis, etc.) needs to contend with its organization into chromatin (the protein-DNA complex that is present within the nucleus of eukaryotic cells).
      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.
      Why do you have to shear the DNA down less than 1000 base pairs (to about three nucleosomes ~400-500bp)?To insure good resolution for ChIP. If your average fragment size is greater than 1000 bp, you could be pulling down DNA that contains your target sequence for PCR but the protein of interest may be over 700 nucleotides distant from your target.
      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.

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      Kit Component

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

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      Categories

      Life Science Research > Kits & Assays > ChIP Kits > Kits and Assays
      Life Science Research > Antibodies and Assays > Immunoassays > Immunoprecipitation (IP) > Chromatin Immunoprecipitation (ChIP) > ChIP Kits & Beads