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  • hSMUG1

    Description

    Human single-strand-selective monofunctional uracil-DNA Glycosylase SMUG1 excises deoxyuracil and deoxyuracil-derivatives bearing an oxidized group at C5, such as 5-hydroxyuracil, 5-hydroxymethyluracil and 5-formyluracil in ssDNA and dsDNA (1,2,3).

    Highlights

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

    Product Source

    An E. coli strain which carries the cloned human SMUG1 gene.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    NEBuffer 1-2010X
    BSA-2010 mg/ml

    Properties and Usage

    Unit Definition

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

    Diluent Compatibility: Diluent C

    Reaction Conditions

    1X NEBuffer 1
    Supplement with 100 μg/ml BSA
    Incubate at 37°C

    1X NEBuffer 1:
    10 mM Bis-Tris-Propane-HCl
    10 mM MgCl2
    1 mM DTT
    pH 7 @ 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

    65°C for 20 min

    Molecular Weight

    Theoretical: 29861 daltons

    Unit Assay Conditions

    1X NEBuffer 1 supplemented with 100 µg/ml BSA containing 5 pmol of fluorescently labeled oligonucleotide duplex in a total reaction volume of 10 µl. After release of dU, the oligonucleotide is cleaved by treating the resulting AP site with 100 mM NaOH for 10 minutes at 80°C.

    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. hSMUG1 has 50% activity on 5-hydroxymethyluracil when compared to uracil. hSMUG1 has 50% activity on ssDNA compared to ds DNA.

    References

    1. Cannon-Crlson, S.V. et al. (1989). J. Biol. Chem. 264, 13306-13312.
    2. Masaoka, A. et al. (2003). Biochemistry. 42, 5003-5012.
    3. Wibley, J.E.A. et al. (2003). Cell. 11, 1647-1659.
    4. Singh, N. et al. (1998). Experimental Cell  Research. 175, 184-191.
    5. Collins, A. et al. (1993). Carcinogenesis . 14, 1733-1735. .
    6. Collins, A. et al. (1996). Environmental Health Perspectives. 104, 465-469.

    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 hSMUG1 in the NEBuffers 1-4?
    2. What is the molecular weight of hSMUG1?
    3. What substrate is used to test hSMUG1?
    4. Is hSMUG1 a tagged protein?

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