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Prices are in US dollars and valid only for US orders.
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- Isolated from a recombinant source
- Supplied with 10X Reaction Buffer
Description:
hOGG1 (α isoform) is an 8-oxoguanine DNA glycosylase which acts both as a N-glycosylase and an AP-lyase. The N-glycosylase activity releases damaged purines from double stranded DNA, generating an apurinic (AP) site. The AP-lyase activity cleaves 3´ to the AP site leaving a 5´ phosphate and a 3´-phospho-α, β-unsaturated aldehyde.
Some of the damaged bases recognized and removed by hOGG1 include 7, 8-dihydro-8-oxoguanine (8-oxoguanine) when base paired with cytosine, 8-oxoadenine when base paired with cytosine, foramidopyrimidine (fapy)-guanine and methy-fapy-guanine (1,2).
Source:
An E. coli strain that carries the cloned human ogg1 gene (3).
Applications:- Single cell gel electrophoresis (Comet assay) (4,5,6)
- Alkaline elution (7)
- Alkaline unwinding (8)
Reagents Supplied:
NEBuffer 2 (10X)
BSA (100X)
Enzyme Properties
Heat Inactivation:
160 units at 65°C for 15 minutes
Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer 2
Supplemented with 100 μg/ml Bovine Serum Albumin
Incubate at
37°C.
1X NEBuffer 2:
10 mM Tris-HCl
50 mM NaCl
10 mM MgCl2
1 mM Dithiothreitol
pH 7.9 @ 25°C
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 8-oxoguanine base paired with a cytosine in a total reaction volume of 10 µl in 1 hour at 37°C in 1X NEBuffer 2 containing 10 pmol of fluorescently labeled oligonucleotide duplex.
Unit Assay Conditions:
1XNEBuffer 2 containing 10 pmol of fluorescently labeled oligonucleotide duplex.
Recommended dilution for the Comet Assay:
1:102 to 1:103 (4,5,6,9). See also: http://cometassay.com/materials_and_protocols.htm
Concentration:
1,600 units/ml
Storage Conditions:
20 mM Tris-HCl
50 mM NaCl
1 mM EDTA
200 µg/ml BSA
50% Glycerol
pH 8.0 @ 25°C
Storage Temperature:
-20°C
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of λDNA (HindIII digest)
and 16 units of hOGG1 incubated for 16 hours at 37ºC
resulted in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis.
Exonuclease Activity:
Incubation of a 50 μl reaction containing 8 units of hOGG1 with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (205 cpm/μg) for 4 hours at 37ºC
released < 0.1% of the total radioactivity.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 8 units of hOGG1 with 1 μg of
ΦX174 RF I DNA for 4 hours at 37ºC resulted
in < 5% conversion to RFII as determined by agarose gel electrophoresis.
References
- Bjoras, M. et al. (1997) Opposite base-dependent reactions of a human base excision repair enzyme on DNA containing 7, 8-dihydro-8-oxoguanine and abasic sites. EMBO J., 16, 6314-6322.
- Boiteux, S. and Radicella, J. (1999) Base excision repair of 8-hydroxyguanine protects DNA from endogenous oxidative stress. Biochimie, 81, 59-67.
- Radicella, J., Dherin, C., Desmze, C., Fox, M. and Boiteux, S. (1997) Cloning and characterization of hOGG1, a human homolog of the OGG1 gene of Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA, 94, 8018-8015.
- Singh, N., McCoy, M., Tice, R. and Schneider, L. (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell. Res., 175, 184-191.
- Collins, A., Duthie, S. and Dobson, V. (1993) Direct enzymatic detection of endogenous oxidative base damage in human lymphocyte DNA. Carcinogenesis, 14, 1733-1735.
- Collins, A., Dusinska, M., Gedik, C. and Stetina, R. (1996) Oxidative damage to DNA: do we have a reliable biomarker?. Environ. Health Perspect., 104, 465-469.
- Pflaum, M., Will, O., Mahler, H.-C. and Epe, B. (1998) DNA oxidation products determined with repair endonucleases in mammalian cells: types, basal levels and influence of cell proliferation. Free Rad. Res., 29, 585-594.
- Hartwig, A., Dally, H. and Schlepegrell, R. (1996) Sensitive analysis of oxidative DNA damage in mammalian cells: use of the bacterial Fpg protein in combination with alkaline unwinding. Toxicol. Lett., 88, 85-90.
- Gutherie, E., New England Biolabs, Inc., unpublished observations.
Reagents Sold Separately
NEBuffer 2
BSA
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