17-371 | EZ-ChIP™

17-371
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      Overview

      Key Spec Table

      Description
      Catalogue Number 17-371
      Brand Family Upstate
      Trade Name
      • Upstate
      • EZ-ChIP
      Description EZ-ChIP™
      Overview Chromatin 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.
      References
      Product Information
      Components Elution Reagent C 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
      Presentation Contains 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
      Application Contains all necessary reagents to perform 22 individual chromatin immunoprecipitation (ChIP) reactions.
      Biological Information
      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 Conditions Upon 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
      Packaging Kit capacity: 22 chromatin immunoprecipitation assays
      Qty/Pck 22 assays
      Material Size 22
      Material Package assays
      Transport Information
      Supplemental Information
      Specifications

      Documentation

      Certificates of Analysis

      TitleLot Number
      EZ ChIP™ Chromatin Immunoprecipitation Kit 2455888
      EZ ChIP™ Chromatin Immunoprecipitation Kit 2465173
      EZ ChIP™ Chromatin Immunoprecipitation Kit 2475771
      EZ ChIP™ Chromatin Immunoprecipitation Kit 2474931
      EZ ChIP™ Chromatin Immunoprecipitation Kit - 2329783 2329783
      EZ ChIP™ Chromatin Immunoprecipitation Kit - 2382613 2382613
      EZ ChIP™ Chromatin Immunoprecipitation Kit - 2388865 2388865
      EZ ChIP™ Chromatin Immunoprecipitation Kit - 2391138 2391138
      EZ ChIP™ Chromatin Immunoprecipitation Kit - 2424117 2424117
      EZ ChIP™ Chromatin Immunoprecipitation Kit - 2428703 2428703
      EZ ChIP™ Chromatin Immunoprecipitation Kit - 2436402 2436402
      EZ ChIP™ Chromatin Immunoprecipitation Kit - 2451404 2451404
      EZ ChIP™ Chromatin Immunoprecipitation Kit - 2453188 2453188
      EZ ChIP™ Chromatin Immunoprecipitation Kit - 2455887 2455887
      EZ ChIP™ Chromatin Immunoprecipitation Kit - 2294334 2294334
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - 2135701 2135701
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - 1959303 1959303
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - 1982652 1982652
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - 1993874 1993874
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - 1999621 1999621
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - 2003364 2003364
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - 2014530 2014530
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - 2025132 2025132
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - 2029679 2029679
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - 2032848 2032848
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - 2041449 2041449
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - 2048872 2048872
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - 2069527 2069527
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - 2113313 2113313
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - 2178405 2178405
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - 2196009 2196009
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - 2212023 2212023
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - 2225259 2225259
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - 2227409 2227409
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - 2251947 2251947
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - 2264636 2264636
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      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - 46020 46020
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - DAM1400036 DAM1400036
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - DAM1413895 DAM1413895
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - DAM1423751 DAM1423751
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - DAM1524127 DAM1524127
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - DAM1556786 DAM1556786
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - DAM1574092 DAM1574092
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - DAM1604878 DAM1604878
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - DAM1640615 DAM1640615
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - DAM1658637 DAM1658637
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - DAM1673555 DAM1673555
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - DAM1673558 DAM1673558
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - DAM1708226 DAM1708226
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - DAM1718541 DAM1718541
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - DAM1732839 DAM1732839
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - DAM1745423 DAM1745423
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - DAM1764547 DAM1764547
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - DAM1780440 DAM1780440
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - DAM1807996 DAM1807996
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - DAM1817732 DAM1817732
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - DAM1834062 DAM1834062
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - JBC1870038 JBC1870038
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - JBC1881386 JBC1881386
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - JBC1900565 JBC1900565
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - JBC1915870 JBC1915870
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - JBC1920643 JBC1920643
      Millipore - COA - EZ ChIP Chromatin Immunoprecipitation Kit - JBC1925537 JBC1925537
      Millipore - COA - EZ ChIP. Chromatin Immunoprecipitation Kit - JBC1369109 JBC1369109
      Millipore - COA - EZ ChIP. Chromatin Immunoprecipitation Kit - JBC1386670 JBC1386670
      Millipore - COA - EZ-ChIPtrade; - DAM1411273A DAM1411273A
      Millipore - COAs - EZ-ChIPtrade; - 0605030944 0605030944
      Millipore - COAs - EZ-ChIPtrade; - 0606034009 0606034009
      Millipore - COAs - EZ-ChIPtrade; - 0608038775 0608038775
      Millipore - COAs - EZ-ChIPtrade; - 0609040497 0609040497
      Millipore - COAs - EZ-ChIPtrade; - 0611045414 0611045414
      Millipore - COAs - EZ-ChIPtrade; - 0612047433 0612047433
      Millipore - COAs - EZ-ChIPtrade; - 0701049641 0701049641
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      Millipore - COAs - EZ-ChIPtrade; - 31689 31689
      Millipore - COAs - EZ-ChIPtrade; - 31759 31759
      Millipore - COAs - EZ-ChIPtrade; - 32035 32035
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      Millipore - COAs - EZ-ChIPtrade; - 33461 33461
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      Millipore - COAs - EZ-ChIPtrade; - 46011 46011
      Millipore - COAs - EZ-ChIPtrade; - 46012 46012

      References | 37 Available | See All References

      Reference overviewApplicationSpeciesPub Med ID
      The Atypical Histone MacroH2A1.2 Interacts with HER-2 Protein in Cancer Cells.
      Xiufen Li,Jinqiu Kuang,Yi Shen,Martin M Majer,Chad C Nelson,Krishna Parsawar,Karen A Heichman,Scott K Kuwada
      The Journal of biological chemistry 287 2012

      Show Abstract
      22589551
      Hypoxia upregulates CD147 through a combined effect of HIF-1α and Sp1 to promote glycolysis and tumor progression in epithelial solid tumors.
      Xia Ke,Fei Fei,Yanke Chen,Li Xu,Zheng Zhang,Qichao Huang,Hongxin Zhang,Hushan Yang,Zhinan Chen,Jinliang Xing
      Carcinogenesis 33 2012

      Show Abstract
      22678117
      Malat1 is not an essential component of nuclear speckles in mice.
      Shinichi Nakagawa,Joanna Y Ip,Go Shioi,Vidisha Tripathi,Xinying Zong,Tetsuro Hirose,Kannanganattu V Prasanth
      RNA (New York, N.Y.) 18 2012

      Show Abstract
      22718948
      Onecut transcription factors act upstream of Isl1 to regulate spinal motoneuron diversification.
      Agnès Roy,Cédric Francius,David L Rousso,Eve Seuntjens,Joke Debruyn,Georg Luxenhofer,Andrea B Huber,Danny Huylebroeck,Bennett G Novitch,Frédéric Clotman
      Development (Cambridge, England) 139 2012

      Show Abstract
      22833130
      E2F7 and E2F8 promote angiogenesis through transcriptional activation of VEGFA in cooperation with HIF1.
      Bart G M W Weijts,Walbert J Bakker,Peter W A Cornelissen,Kuo-Hsuan Liang,Frank H Schaftenaar,Bart Westendorp,Charlotte A C M T de Wolf,Maya Paciejewska,Colinda L G J Scheele,Lindsey Kent,Gustavo Leone,Stefan Schulte-Merker,Alain de Bruin
      The EMBO journal 31 2012

      Show Abstract
      22903062
      ATF4 orchestrates a program of BH3-only protein expression in severe hypoxia.
      Luke R G Pike,Kanchan Phadwal,Anna Katharina Simon,Adrian L Harris
      Molecular biology reports 39 2012

      Show Abstract
      23090478
      PALB2 interacts with KEAP1 to promote NRF2 nuclear accumulation and function.
      Jianglin Ma,Hong Cai,Tongde Wu,Bijan Sobhian,Yanying Huo,Allen Alcivar,Monal Mehta,Ka Lung Cheung,Shridar Ganesan,Ah-Ng Tony Kong,Donna D Zhang,Bing Xia
      Molecular and cellular biology 32 2012

      Show Abstract
      22331464
      Epigenetic modulation of the protein kinase A RIIα (PRKAR2A) gene by histone deacetylases 1 and 2 in human smooth muscle cells.
      Magdalena Karolczak-Bayatti,Andrew D Loughney,Stephen C Robson,G Nicholas Europe-Finner
      Journal of cellular and molecular medicine 15 2011

      Show Abstract
      19818097
      Histone Modifications on the adrenergic induction of type iI deiodinase in rat pinealocytes.
      Chik CL, Price DM, Ho AK
      Molecular and cellular endocrinology 2011

      Show Abstract
      21704117
      H3K4 tri-methylation provides an epigenetic signature of active enhancers.
      Aleksandra Pekowska,Touati Benoukraf,Joaquin Zacarias-Cabeza,Mohamed Belhocine,Frederic Koch,Hélène Holota,Jean Imbert,Jean-Christophe Andrau,Pierre Ferrier,Salvatore Spicuglia
      The EMBO journal 30 2011

      Show Abstract
      21847099

      Brochure

      Title
      Product Selection Guide: Shaping Epigenetics Discovery

      Data Sheet

      Title
      Reprogramming Cell Fate and Function Novel Strategies for iPSC Generation, Characterization, and Differentiation

      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.
      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.
      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.
      From where are the primer sequences derived for the kit?The primer sequences are based on the Human GAPDH promoter. The GenBank number is NT_009759.15, using nts:6497145-6498136.
      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.
      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.
      What are the best primer designs?Primer length should be 24 nucleotides; they should have 50% GC content, and a Tm of 60°C. Don't try to amplify anything larger than 600-800 nucleotides. Attempt to stay away from sequences that are not unique within the genome.
      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.
      Can I use this kit with transcription factors?Yes, but the antibodies must be very good and generally you will want to increase the fixation times to insure maximal cross-linking.
      Why do some antibodies work for ChIP and others don't?For ChIP to be successful, it requires that the epitope recognized by the antibody that you are employing be available after cross-linking and not buried in the protein complex. Antibodies also have to be of very high avidity so that the interaction with the protein will survive the washing steps. It is also important that the protein you are trying to IP will cross-link efficiently to the chromatin.
      What is the difference between the 'Chip Grade' and 'Non-Chip Grade' antibodies?Nothing really. Many customers perceived that only 'ChIP grade' antibodies would work in ChIP so we have eliminated this term. 'ChIP grade' antibodies were designed specifically for ChIP, but also work well in other applications. It is best to consult the list of tested applications for each product to determine if it is suitable for use in ChIP.
      Can I use plant samples with the ChIP kit?Although ChIP kits have been used mostly in mammalian samples, it is possible that some antibodies will recognize plant Histone due to high sequence homology. However, you may need to alter the extraction/lysis buffer. Plant preparation, lysis buffer, and sonication strategies, to shear plant Histones down to about three nucleosomes, would have to be worked out.

      Please see the following references on Histone acetylation and synthesis in plants:
      Wang, et al.(2002), The Plant Journal 32 (5), 831-843
      Waterborg, J.H. Biochem. Cell Biol. 80: 363-378, 2002
      Waterborg, J.H. and Kapros, Cell Biol. 80: 279-293, 2002
      Waterborg, J.H. JBC, 268: 4912-4917, 1993
      Waterborg. J.H. JBC, 268: 4918-4921, 1993
      Waterborg, J.H. JBC, 265: 17157-17161, 1990
      Waterborg, J.H. Plant Physiol. 96: 453-458, 1991
      Waterborg, J.H. Plant Mol. Biol. 18: 181-187, 1992
      Waterborg, J.H. Biotechnology Quarterly 9: 12-13, 1992
      Waterborg, J.H. Biochemistry 31: 6211-6219, 1992
      Can I use tissue samples with the ChIP kit?You can, please consult this protocol for additional information (This protocol is based upon protocols from Mark Biggin, Dave Allis and Richard Treisman) This protocol has successfully been used on livers, spleens, colons and whole mouse embryos. However, you may have to optimize conditions for your specific tissue type: Day 1: 1. Chop tissue into small pieces with a razor blade or scalpel. Transfer tissue into a tube with a screw cap lid and add a small (5-10mls) amount of tissue culture media. We have not noticed a difference with different types of media. 1X PBS should also work. Add formaldehyde to a final concentration of 1% and rotate tube at room temperature for 15 minutes. We generally use 0.03g of tissue per antibody. The exact amount of tissue that you will need depends upon how abundant your protein is, how strongly your antibody binds and how efficient your cross linking is. 2. Stop the cross linking reaction by adding glycine to a final concentration of 0.125 M. Continue to rotate at room temp for 5 minutes. 3. Centrifuge samples at low speed, decant media and wash once with cold 1X PBS. Centrifuge. Add a small (1-2mls per sample) amount of cold 1X PBS and disaggregate tissues. For this step, we use a Medimachine from Becton Dickinson. However, a dounce homogenizer also works. Centrifuge at low speed to pellet cells and decant supernatant. 4. Resuspend cell pellet in cell lysis buffer plus the protease inhibitors PMSF (10 ul per ml), aprotinin (1 ul per ml) and leupeptin (1 ul per ml). The final volume of cell lysis buffer should be sufficient so that there are no clumps of cells. Incubate on ice for 10-15 minutes. Cells can also be dounced on ice with a B dounce several times to aid in nuclei release. 5. Microfuge at 5,000 rpm for 5 minutes at 40 C to pellet the nuclei. 6. Resuspend nuclei in nuclei lysis buffer plus the same protease inhibitors as the cell lysis buffer. Incubate on ice for 10-20 minutes. 7. Add
      Why does the kit contain low and high salt buffers?As a gradual increase in stringency occurs, chromatin will be prevented from interacting with the beads. Unlike DNA hybridization, where lower salt equates with higher stringency, in the Chromatin IP, higher salt leads to higher stringency washing.
      At what point can I stop and freeze my samples?There are several points at which the procedure may be put on hold.
      A) After pelleting cells post formaldehyde and washes, you can freeze the cell pellet at -70° C.
      B) After making the lysate you can freeze at -70° C.
      C) After sonication, you can store the chromatin by aliquoting and storing at -70° C.
      D) After you reverse the cross-links prior to DNA purification you may store at -20° C.
      What is the purpose/chemistry behind the use of LiCl and NaCl in the wash buffers?The use of different types of salts improves the effective removal of non-specific chromatin interactions with the agarose beads. Lithium is also soluble in buffers containing high amounts of SDS.
      Can I alter the time and temperature of cross-link reversal?Reversal of cross-links for shorter than four hours is not recommended. However, it is possible to leave the samples at 65° C overnight for cross-link reversal provided steps are taken to insure that the samples do not dry out.
      At times, when I try to pellet my beads, they don't form a tight pellet and it is difficult to remove the supernatant. This happens even after I spin longer and/or faster. What can I do the remedy this?High-speed centrifugation is not necessary to pellet agarose beads. Often high-speed centrifugation will result in breaking agarose into smaller beads called 'fines'. If you are having trouble removing the supernatant from your pellet, you could use a small gel loading tip or a needle and syringe. This will allow you to remove the supernatant without disrupting the pellet.

      User Guides

      Title
      EZ-ChIP™ Chromatin Immunoprecipitation Kit