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  • Endonuclease IV

    Description

    Endonuclease IV can act on a variety of oxidative damage in DNA (1). The enzyme is apurinic/apyrimidinic (AP) endonuclease that will hydrolyse intact AP sites in DNA. AP sites are cleaved at the first phosphodiester bond that is 5´ to the lesion leaving a hydroxyl group at the 3´ terminus and a deoxyribose 5´-phosphate at the 5´ terminus. The enzyme also has a 3´ -diesterease activity and can release phosphoglycoaldehyde, intact deoxyribose 5-phosphate and phosphate from the 3´ end of DNA.


    Highlights

    • Isolated from a recombinant source
    • Supplied with 10X Reaction Buffer

    Product Source

    An E.coli strain which carries the cloned Endo IV gene.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    NEBuffer 3-2010X

    Advantages and Features

    Applications

    • Single cell gel electrophoresis (Comet assay)(2,3,4)
    • Alkaline elution (5)
    • Alkaline unwinding (6)

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme required to cleave 1 pmol of a 34-mer oligonucleotide duplex containing a single AP site* in a total reaction volume of 10 µl in 1 hour at 37°C.

    *An AP site is created by treating 10 pmol of a 34 mer oligonucleotide duplex containing a single uracil residue with 1 unit of Uracil-DNA Glycosylase (UDG) for 2 minutes at 37°C.

    Reaction Conditions

    1X NEBuffer 3
    Incubate at 37°C

    1X NEBuffer 3:
    100 mM NaCl
    50 mM Tris-HCl
    10 mM MgCl2
    1 mM DTT
    pH 7.9 @ 25°C

    Storage Temperature

    -20°C

    Storage Conditions

    10 mM Tris-HCl
    250 mM NaCl
    1 mM DTT
    0.1 mM EDTA
    200 μg/ml BSA
    50% Glycerol
    0.15% Triton® X-100
    pH 7.4 @ 25°C

    Heat Inactivation

    85°C for 20 min

    Unit Assay Conditions

    1X NEBuffer 3 containing 10 pmol of fluorescently labeled oligonucleotide duplex in a total reaction volume of 10 μl.

    Quality Control

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Endonuclease Activity (Nicking):
      The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
    • Exonuclease Activity (Radioactivity Release):
      The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
    • Non-Specific DNase Activity (16 hour):
      The product is tested for non-specific nuclease degradation in a reaction containing a DNA substrate. After incubation for 16 hours there is no detectable degradation of the DNA substrate as determined by agarose gel electrophoresis.
    • Protein Purity (SDS-PAGE):
      The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest band in a sample of known dilution. The purity is determined by SDS-PAGE.

    Notes

    1. Recommended Dilution for the Comet Assay: 1.104 to 1:105(2,3,4,7). For a protocol please visit: http://cometassay.com

    References

    1. Levin, J. et al. (1988). J. Gen. Physiol. . 33, 349-362.
    2. Singh, N. et al. (1961). Experimental Cell  Research . 175, 184-191.
    3. Collins, A. etal. (1993). Carcinogenesis . 14, 1733-1735.
    4. Collins, A. et al. (1996). Environmental Health Perspectives . 104, 465-469.
    5. Pflaum, M. et al. (1998). Free Rad. Res. 29, 585-594.
    6. Hartwig, A. et al. (1996). Toxicology Letters. 88, 85-90.
    7. Marks, K., New England Biolabs, Inc. unpublished observations.

    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. What is the activity of Endonuclease IV in the NEBuffers?
    2. Does Endonuclease IV work in Thermopol buffer?
    3. What is the molecular weight for Endonuclease IV?
    4. Does Endonuclease IV cleave ssDNA?
    5. Will the 5' terminus left by an Endonuclease IV cleavage be suitable for subsequent ligation by T4 DNA ligase?
    6. In addition to AP sites, does Endonuclease IV have cleavage activity on other types of DNA damage?
    7. Can an abasic site be created and then cleaved with Endonuclease IV in the same reaction? How active is Uracil-DNA-Glycosylase (cat# M0280S) in Endonuclease IV buffer?

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