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  • hNEIL1

    Discontinued Date

    07/02/2012
    cloned at neb recombinant unique buffer incubation temp heat inactivation
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    Discontinued Products

    Description

    Human NEIL1 acts as both an N-glycosylase and an AP-lyase. The N-glycosylase activity releases oxidized pyrimidines and purines from double-stranded DNA, generating an apurinic (AP site). The AP-lyase activity cleaves 3´ and 5´ to the AP site leaving a 5´ phosphate and a 3´ phosphate. Damaged bases recognized and removed by hNEIL1 include thymine glycol, 5,6-dihydrothymine, 5,6-dihydroxyuracil, 5-hydroxycytosine, 5-hydroxyuracil, 5-formyluracil, 8oxo-guanine opposite C, T or G, 4,6-diamino-5-formamidopyrimidine and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (1,2).

    Product Source

    An E. coli strain that carries the cloned human NEIL1 gene.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    Endonuclease VIII Reaction Buffer10X

    Advantages and Features

    Applications

    • Oxidative DNA damage studies
    • Single cell gel electrophoresis (comet assay) (4,5,6)

    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. 

    Diluent Compatibility: Diluent Buffer A
    50 mM KCl, 10 mM Tris-HCl, 0.1 mM EDTA, 1 mM DTT, 200 µg/ml BSA and 50% glycerol (pH 7.4 @ 25°C)

    Reaction Conditions

    1X Endonuclease VIII Reaction Buffer
    Incubate at 37°C

    1X Endonuclease VIII Reaction Buffer:
    10 mM Tris-HCl
    75 mM NaCl
    1 mM EDTA
    pH 8 @ 25°C

    Usage Concentration

    1%

    Dilution for Comet Assay

    Storage Temperature

    -20°C

    Storage Conditions

    10 mM Tris-HCl
    50 mM KCl
    1 mM DTT
    0.1 mM EDTA
    200 μg/ml BSA
    50% Glycerol
    pH 7.4 @ 25°C

    Heat Inactivation

    65°C for 20 min

    Molecular Weight

    Theoretical: 43685 daltons

    Unit Assay Conditions

    1X Endonuclease VIII Reaction Buffer containing 5 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. Compared to the activity on AP-site, hNEIL1 has ≥ 50% activity on thymine glycol and ≥ 25% activity on dhU, dhT, 5hoU and 5hoC. hNEIL1 is active on 8oxoG opposite C,T and G, but is not active on an 8oxoG:A base pair. hNEIL1 is not active on 5-hydroxymethyluracil.
    2. Oxidative DNA damage studies
    3. Single cell gel electrophoresis (comet assay) (3,4,5)

    References

    1. Hazra, T.K. et al. (2002). Proc. Natl. Acad. Sci. 99
    2. Bandaru, V. et al. (2002). DNA Repair. 1
    3. Singh, N., McCoy, M., Tice, R. and Schneider, L. (1988). Experimental Cell Research. 175
    4. Collins, A., Duthie, S. and Dobson, V. (1993). Carcinogenesis. 14
    5. Collins, A., Dusinska, M., Gedik, C. and Stetina, R. (1996). Environmental Health Perspectives. 104
    6. Katafuchi, A. et al. (2004). J. Biol. Chem. 14

    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. NEB's data card for hNEIL1 states that this enzyme does not cleave 5-hmU containing substrates. However, there are two publications by the Qiu-Mei Zhang group from Kyoto University demonstrating cleavage of 5-hmU oligos by both hNEIL1 and Nei, the E.coli homolog. Can you perhaps shed some light on this matter?
    2. What is the activity of hNEIL1 in NEBuffers?
    3. What is the Molecular Weight of hNEIL1?
    4. What is the substrate used to test hNEIL1?
    5. Is hNEIL1 a tagged protein?

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