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  • hAAG

    Description

    Human Alkyladenine DNA Glycosylase (hAAG) excises alkylated and oxidative DNA damaged sites, including 3-methyladenine, 7-methylguanine, 1,N6-ethenoadenine and hypoxanthine. hAAG catalyzes the hydrolysis of the N-glycosidic bond to release the damaged base. hAAG is also known as methylpurine DNA glycosylase (MPG) (1,2,3) or 3-methyladenine-DNA glycosylase (ANPG) (4).


    Product Source

    An E. coli strain which carries the cloned truncated human AAG gene (1)

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    ThermoPol® Reaction Buffer-2010X

    Advantages and Features

    Applications

    • Single cell gel electrophoresis (Comet Assay)(5,6,7) 
    • Alkaline elution (8)
    • Alkaline unwinding (9)

    Properties and Usage

    Unit Definition

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

    Reaction Conditions

    1X ThermoPol® Reaction Buffer
    Incubate at 37°C

    1X ThermoPol® Reaction Buffer:
    20 mM Tris-HCl
    10 mM (NH4)2SO4
    10 mM KCl
    2 mM MgSO4
    0.1% Triton® X-100
    pH 8.8 @ 25°C

    Storage Temperature

    -20°C

    Storage Conditions

    10 mM Tris-HCl
    100 mM KCl
    1 mM DTT
    0.1 mM EDTA
    50% Glycerol
    0.5% Tween® 20
    0.5% NP-40

    Heat Inactivation

    65°C for 20 min

    Molecular Weight

    Theoretical: 25752 daltons

    Unit Assay Conditions

    1X ThermoPol 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.

    References

    1. O'Brien, P. and Ellenberger, T. (2003). Biochemistry. 42, 12418-12429.
    2. Abner, C.W.et al. (2001). J. Biol. Chem.. 276, 13379-13387.
    3. Samson, L. (1991). Proc. Natl. Acad.. Sci. 88, 9127-9131.
    4. Lau, A.Y. et al. (1998). Cell. 95, 249-258.
    5. Singh, N. et al. (1961). Experimental Cell  Research. 175, 184-191.
    6. Collins, A. et al. (1993). Carcinogenesis . 14, 1733-1735.
    7. Collins, A. et al. (1996). Environmental Health Perspectives. 104, 465-469.
    8. Pflaum, M. et al. (1996). Free Rad. Res. 29, 585-594.
    9. Hartwig, A. et al. (1996). Toxicology Letters.. 88, 85-90.

    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 substrate is used to test hAAG?
    2. What is the molecular weight of hAAG?
    3. Is hAAG a tagged protein?

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