17-371 EZ-ChIP™

17-371
22 assays  Kit capacity: 22 chromatin immunoprecipitation assays
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      Overview

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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 Number17-371
      Trade Name
      • EZ-ChIP
      DescriptionEZ-ChIP™
      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:
      Easier: Spin columns make DNA purification easier and more reliable - no more messy phenol-chloroform extractions.
      Quicker: All reagents to process your samples are included - you don't have to spend valuable time making them.
      Greater Reproducibility: Positive and negative control antibodies and PCR primers are included to help validate your results and to troubleshoot your experiments.
      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
      • DNA Purification Spin Columns and Collection Tubes
      • ChIP Blocked Protein G Agarose
      • Anti-RNA Polymerase II
      • Control PCR Primers
      • Normal Mouse IgG
      • Bind, Wash and Elution Reagents
      • Protease Inhibitor Cocktail II
      • RNase A
      • Proteinase K
      • All required buffers
      • ChIP Dilution Buffer
      • Low Salt Immune Complex Wash Buffer
      • High Salt Immune Complex Wash Buffer
      • LiCl Immune Complex Wash Buffer
      • TE Buffer
      • 0.5M EDTA
      • 5M NaCl
      • SDS Lysis Buffer
      • 1M Tris-HCl, pH 6.5
      • 10X PBS
      • 10X Glycine
      • 1M NaHCO3
      • Control Primers
      • 20% SDS
      • Spin Filters
      • Collection Tubes
      • Bind Reagent A
      • Wash Reagent B
      • Elution Reagent C
      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
      ApplicationThis EZChIP kit contains all necessary reagents to perform 22 individual chromatin immunoprecipitation (ChIP) reactions using inexpensive protein G agarose 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

      EZ-ChIP™ SDS

      Title

      Safety Data Sheet (SDS) 

      EZ-ChIP™ Certificates of Analysis

      TitleLot Number
      EZ ChIP Chromatin Immunoprecipitation Kit - 21357012135701
      EZ ChIP Chromatin Immunoprecipitation Kit - 19593031959303
      EZ ChIP Chromatin Immunoprecipitation Kit - 19826521982652
      EZ ChIP Chromatin Immunoprecipitation Kit - 19938741993874
      EZ ChIP Chromatin Immunoprecipitation Kit - 19996211999621
      EZ ChIP Chromatin Immunoprecipitation Kit - 20033642003364
      EZ ChIP Chromatin Immunoprecipitation Kit - 20145302014530
      EZ ChIP Chromatin Immunoprecipitation Kit - 20251322025132
      EZ ChIP Chromatin Immunoprecipitation Kit - 20296792029679
      EZ ChIP Chromatin Immunoprecipitation Kit - 20328482032848

      References

      Reference overviewApplicationSpeciesPub Med ID
      A homologue of Nr5a1 activates cyp19a1a transcription additively with Nr5a2 in ovarian follicular cells of the orange-spotted grouper.
      Shi, B; Lu, H; Zhang, L; Zhang, W
      Mol Cell Endocrinol  460  85-93  2018

      Show Abstract
      28694164 28694164
      Inhibition of neddylation by MLN4924 improves neointimal hyperplasia and promotes apoptosis of vascular smooth muscle cells through p53 and p62.
      Ai, TJ; Sun, JY; Du, LJ; Shi, C; Li, C; Sun, XN; Liu, Y; Li, L; Xia, Z; Jia, L; Liu, J; Duan, SZ
      Cell Death Differ  25  319-329  2018

      Show Abstract
      29027989 29027989
      EZH2 regulates dental pulp inflammation by direct effect on inflammatory factors.
      Hui, T; A, P; Zhao, Y; Yang, J; Ye, L; Wang, C
      Arch Oral Biol  85  16-22  2018

      Show Abstract
      29028630 29028630
      Clock represses preadipocytes adipogenesis via GILZ.
      Zhu, Z; Xu, L; Cai, T; Yuan, G; Sun, N; Lu, C; Qian, R
      J Cell Physiol  233  6028-6040  2018

      Show Abstract
      29278648 29278648
      Long non-coding RNA DLEU1 predicts poor prognosis of gastric cancer and contributes to cell proliferation by epigenetically suppressing KLF2.
      Li, X; Li, Z; Liu, Z; Xiao, J; Yu, S; Song, Y
      Cancer Gene Ther  25  58-67  2018

      Show Abstract
      29282356 29282356
      WT1 protein is cleaved by caspase-3 in apoptotic leukemic cells.
      Ruan, J; Gao, S; Yang, J; Li, H; Huang, H; Zheng, X
      Leuk Lymphoma  59  162-170  2018

      Show Abstract
      28395566 28395566
      Metabolic Syndrome Induces Over Expression of the Human AT1R: A Haplotype-Dependent Effect With Implications on Cardio-Renal Function.
      Jain, S; Puri, N; Rana, A; Sirianni, N; Mopidevi, B; Kumar, A
      Am J Hypertens  31  495-503  2018

      Show Abstract
      29036458 29036458
      PAX9 regulates squamous cell differentiation and carcinogenesis in the oro-oesophageal epithelium.
      Xiong, Z; Ren, S; Chen, H; Liu, Y; Huang, C; Zhang, YL; Odera, JO; Chen, T; Kist, R; Peters, H; Garman, K; Sun, Z; Chen, X
      J Pathol  244  164-175  2018

      Show Abstract
      29055049 29055049
      PTEN Is Fundamental for Elimination of Leukemia Stem Cells Mediated by GSK126 Targeting EZH2 in Chronic Myelogenous Leukemia.
      Zhou, J; Nie, D; Li, J; Du, X; Lu, Y; Li, Y; Liu, C; Dai, W; Wang, Y; Jin, Y; Pan, J
      Clin Cancer Res  24  145-157  2018

      Show Abstract
      29070525 29070525
      Effect of long non-coding RNA PVT1 on cell proliferation and migration in melanoma.
      Chen, L; Ma, D; Li, Y; Li, X; Zhao, L; Zhang, J; Song, Y
      Int J Mol Med  41  1275-1282  2018

      Show Abstract
      29286144 29286144

      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.
      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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      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