17-409 | EZ-Magna ChIP™ G - Chromatin Immunoprecipitation Kit

17-409
22 assays  Kit capacity: 22 chromatin immunoprecipitation 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 Number17-409
      Trade Name
      • Magna ChIP
      DescriptionEZ-Magna ChIP™ G - Chromatin Immunoprecipitation Kit
      OverviewChromatin Immunoprecipitation (ChIP) is an important technique allowing the researcher to analyze in vivo interactions of proteins with genomic DNA. Any chromatin-associated or DNA binding protein can be analyzed with this technique, provided a good antibody to the protein exists. One can measure different proteins localized to a specific region of the genome, or the genome wide distribution of a specific protein. Another powerful application of this technique is to analyze changes in histone modifications that correlate with processes like transcription, mitosis or DNA repair.

      Features & Benefits:
      Faster: Magnetic protein G beads allow for the entire ChIP protocol to be done in as little as a day! All reagents to process your samples are included - you don't have to spend valuable time making them.
      Easier: Spin columns make DNA purification easier and more reliable - no more messy phenol-chloroform extractions.
      Greater Reproducibility: Positive and negative control antibodies and PCR primers are included to help validate your results and to troubleshoot your experiments.
      Alternate Names
      • Magnetic 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.
      Materials Required but Not DeliveredMagna Grip™ Rack 8 well ( 20-400) (Now Available!) or similar magnetic rack.
      References
      Product Information
      Components
      • Magnetic Protein G Beads
      • ChIP Dilution Buffer
      • Low Salt Wash Buffer
      • High Salt Wash Buffer
      • LiCl Wash Buffer
      • TE Buffer
      • Cell Lysis Buffer
      • Nuclear Lysis Buffer
      • ChIP Elution Buffer (w/o Proteinase K)
      • 10X Glycine
      • 10X PBS
      • Protease Inhibitor Cocktail II
      • Proteinase K
      • Control Primers
      • Anti-RNA Polymerase II
      • Normal Mouse IgG
      • Spin Filters
      • Collection Tubes
      • Bind Reagent A
      • Wash Reagent B
      • Elution Reagent C
      PresentationTwo boxes containing 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).
      Applications
      ApplicationSingle day chromatin immunoprecipitation (ChIP) kit containing all necessary reagents to perform 22 individual chromatin immunoprecipitation (ChIP) reactions using magnetic G beads. Control primers included.
      Biological Information
      Analytes Available
      • Protein G
      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
      Storage ConditionsUpon receipt, store components at the temperatures indicated on the labels. Kit components are stable for 1 year from date of shipment when stored as directed.
      Packaging Information
      Material Size22 assays
      Material PackageKit capacity: 22 chromatin immunoprecipitation assays
      Transport Information
      Supplemental Information
      Specifications

      Documentation

      SDS

      Title

      Safety Data Sheet (SDS) 

      Certificates of Analysis

      TitleLot Number
      EZ-Magna ChIP G Chromatin Immunoprecipitation Kits - DAM1439048 DAM1439048
      EZ-Magna ChIPtrade; G Chromatin Immunoprecipitation Kit - DAM1421808 DAM1421808
      EZ-Magna ChIPtrade; G Chromatin Immunoprecipitation Kit - DAM1432917 DAM1432917
      EZ-Magna ChIPtrade; G Chromatin Immunoprecipitation Kit - R0708G0032 R0708G0032
      Magna ChIP G or EZ-Magna ChIP G Chromatin Immunoprecipitation Kits - 1963130 1963130
      Magna ChIP G or EZ-Magna ChIP G Chromatin Immunoprecipitation Kits - 1982676 1982676
      Magna ChIP G or EZ-Magna ChIP G Chromatin Immunoprecipitation Kits - 2029683 2029683
      Magna ChIP G or EZ-Magna ChIP G Chromatin Immunoprecipitation Kits - 2032859 2032859
      Magna ChIP G or EZ-Magna ChIP G Chromatin Immunoprecipitation Kits - 2055184 2055184
      Magna ChIP G or EZ-Magna ChIP G Chromatin Immunoprecipitation Kits - 2055186 2055186

      References

      Reference overviewApplicationPub Med ID
      Host factor PRPF31 is involved in cccDNA production in HBV-replicating cells.
      Kinoshita, W; Ogura, N; Watashi, K; Wakita, T
      Biochem Biophys Res Commun  638-644  2017

      Show Abstract
      27864147 27864147
      Regulation of tubular recycling endosome biogenesis by the p53-MICALL1 pathway.
      Takahashi, Y; Tanikawa, C; Miyamoto, T; Hirata, M; Wang, G; Ueda, K; Komatsu, T; Matsuda, K
      Int J Oncol  724-736  2017

      Show Abstract
      28714518 28714518
      Kaiso protects human umbilical vein endothelial cells against apoptosis by differentially regulating the expression of B-cell CLL/lymphoma 2 family members.
      Xue, X; Zhang, J; Lan, H; Xu, Y; Wang, H
      Sci Rep  7116  2017

      Show Abstract
      28769046 28769046
      Metformin disrupts malignant behavior of oral squamous cell carcinoma via a novel signaling involving Late SV40 factor/Aurora-A.
      Chen, CH; Tsai, HT; Chuang, HC; Shiu, LY; Su, LJ; Chiu, TJ; Luo, SD; Fang, FM; Huang, CC; Chien, CY
      Sci Rep  1358  2017

      Show Abstract
      28465536 28465536
      EPSIN 3, A Novel p53 Target, Regulates the Apoptotic Pathway and Gastric Carcinogenesis.
      Mori, J; Tanikawa, C; Ohnishi, N; Funauchi, Y; Toyoshima, O; Ueda, K; Matsuda, K
      Neoplasia  185-195  2017

      Show Abstract
      28152424 28152424
      Fine-tuning and autoregulation of the intestinal determinant and tumor suppressor homeobox gene CDX2 by alternative splicing.
      Balbinot, C; Vanier, M; Armant, O; Nair, A; Penichon, J; Soret, C; Martin, E; Saandi, T; Reimund, JM; Deschamps, J; Beck, F; Domon-Dell, C; Gross, I; Duluc, I; Freund, JN
      Cell Death Differ  2173-2186  2017

      Show Abstract
      28862703 28862703
      SMAD2 Inactivation Inhibits CLDN6 Methylation to Suppress Migration and Invasion of Breast Cancer Cells.
      Lu, Y; Wang, L; Li, H; Li, Y; Ruan, Y; Lin, D; Yang, M; Jin, X; Guo, Y; Zhang, X; Quan, C
      Int J Mol Sci  2017

      Show Abstract
      28867761 28867761
      Identification of a p53 target, CD137L, that mediates growth suppression and immune response of osteosarcoma cells.
      Tsuda, Y; Tanikawa, C; Miyamoto, T; Hirata, M; Yodsurang, V; Zhang, YZ; Imoto, S; Yamaguchi, R; Miyano, S; Takayanagi, H; Kawano, H; Nakagawa, H; Tanaka, S; Matsuda, K
      Sci Rep  10739  2017

      Show Abstract
      28878391 28878391
      Identification of a novel p53 target, COL17A1, that inhibits breast cancer cell migration and invasion.
      Yodsurang, V; Tanikawa, C; Miyamoto, T; Lo, PHY; Hirata, M; Matsuda, K
      Oncotarget  55790-55803  2017

      Show Abstract
      28915553 28915553
      Loss of ULK1 increases RPS6KB1-NCOR1 repression of NR1H/LXR-mediated Scd1 transcription and augments lipotoxicity in hepatic cells.
      Sinha, RA; Singh, BK; Zhou, J; Xie, S; Farah, BL; Lesmana, R; Ohba, K; Tripathi, M; Ghosh, S; Hollenberg, AN; Yen, PM
      Autophagy  169-186  2017

      Show Abstract
      27846372 27846372

      FAQ

      QuestionAnswer
      How many PCR reactions can be done with this kit?There are enough primers and PCR buffer for 4 reactions per IP assuming a 20 microliter volume and assuming the primers are at the recommended concentration as stated in the manual.
      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.
      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.

      User Guides

      Title
      EZ-Magna ChIP™ G

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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