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  • EpiMark® Nucleosome Assembly Kit

    Description

    The EpiMark® Nucleosome Assembly Kit is used to make unmodified recombinant human nucleosomes with user-supplied DNA or the provided control DNA. Purified recombinant human Histone H2A/H2B Dimer and Histone H3.1/H4 Tetramer are mixed with DNA at 2 M NaCl. To generate nucleosomes, the salt concentration is lowered by dilution or dialysis allowing each histone tetramer to associate with two histone dimers and form the histone octamer on the DNA (1,2). A method for assaying nucleosome formation by gel shift assay is included. These nucleosomes may serve as better substrates for enzymes that are inactive on the DNA or one of the core histones alone (3,4). Each reaction creates nucleosomes from ~1 µg of 208 bp DNA and may be scaled depending on the experiment.

    Nucleosome formation.
    A gel shift assay allows visualization of complex formation when various ratios of Octamer* to DNA are used to form mononucleosomes using the provided nucleosome control DNA. When assembly reactions are run on 6% polyacrylamide gel in 0.5X TBE, a characteristic shift occurs from 208 bp to approximately 700 bp.

    • M: Low Molecular Weight DNA Ladder (NEB #N3233)
    • Lane 1: Nucleosome Control DNA
    • Lane 2: 0.5:1 ratio of Octamer* to DNA
    • Lane 3: 1:1 ratio of Octamer* to DNA.

    *Octamer = 2:1 mix of Histone H2A/H2B Dimer and Histone H3.1/H4 Tetramer.


    Kit Components

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    Nucleosome Control DNA-2010 μM

    Advantages and Features

    Features

    • Highly pure, recombinant system
    • Components stable for one year
    • Pre-formed Histone Dimer and Tetramer complexes simplify Octamer formation
    • Dilution protocol only requires a few hours for assembly

    Applications

    • ChIP assay
    • HAT assay (Histone Acetyltransferase Assay)
    • Enzymatic modification assay, (e.g., methylation)

    Properties and Usage

    Storage Temperature

    -20°C

    References

    1. Kornberg, R.D. (1977). Annu. Rev. Biochem . 46, 931-954. PubMedID: 332067
    2. van Holde, K.E. (1989). Chromatin. 1-497.
    3. Li, Y et al. (2009). Jour. of Biol. Chem. 284, 34283-34295. PubMedID: 19808676
    4. Qi, Hank H. et al. (2010). Nature. 466, 503-507. PubMedID: 20622853
    5. Luger et al. (2004). Methods in Enzymology. 375, 23-62. PubMedID: 14870657

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. Introduction (E5350)
    2. Dilution Assembly Protocol (E5350)
    3. Dialysis Assembly Protocol (E5350)
    4. Gel Shift Assay for EpiMark™ Nucleosome Assembly Kit (E5350)
    5. DNA Concentration Formulas for EpiMark™ Nucleosome Assembly Kit (E5350)
    6. EpiMark™ Nucleosome Assembly Kit (E5350)
    It is critical to always add the Dimer and Tetramer last when preparing the assembly reaction.