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 Pack-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.

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.

FAQs

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

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.

Selection Charts

Usage Guidelines & Tips

Interactive Tools

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. Further information regarding NEB product quality can be found here.
  • 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.

Safety Data Sheet

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

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.