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- Isolated from a recombinant source
- Supplied with 10X Reaction Buffer
Description:
Fpg (formamidopyrimidine [fapy]-DNA glycosylase) (also known as 8-oxoguanine DNA glycosylase) 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 both 3´ and 5´ to the AP site thereby removing the AP site and leaving a 1 base gap. Some of the damaged bases recognized and removed by Fpg include 7, 8-dihydro-8-oxoguanine (8-oxoguanine), 8-oxoadenine, fapy-guanine, methy-fapy-guanine, fapy-adenine, aflatoxin B1-fapy-guanine, 5-hydroxy-cytosine and 5-hydroxy-uracil (1,2).
Source:
An E.coli strain that carries the cloned fpg gene (3)
Applications:- Single cell gel electrophoresis (Comet assay) (4,5,6)
- Alkaline elution (7)
- Alkaline unwinding (8)
- Modified nick translation (9)
Reagents Supplied:
NEBuffer 1
BSA
Enzyme Properties
End Product: 5´ and 3´ phosphate
Heat Inactivation:
160 units at 60°C for 10 minutes
Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer 1
Supplemented with 100 μg/ml Bovine Serum Albumin
Incubate at
37°C.
1X NEBuffer 1:
10 mM Bis-Tris-Propane-HCl
10 mM MgCl2
1 mM Dithiothreitol
pH 7.0 @ 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.
Unit Assay Conditions:1X NEBuffer 1 containing 10 pmol of fluorescently labeled oligonucleotide duplex in a total reaction volumn of 10 μl.
Concentration:
8,000 units/ml
Storage Conditions:
20 mM Tris-HCl
50 mM NaCl
0.5 mM EDTA
200 µg/ml BSA
50% Glycerol
pH 8.0 @ 25°C
Storage Temperature:
-20°C
Notes
Usage notes:- Recommended dilution for the Comet Assay: 1:103 to 1:104 (4,5,6). See also: http://cometassay.com/materials_and_protocols.htm
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 40 units of Fpg 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 40 units of Fpg with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (205 cpm/μg) for 4 hours at 37ºC
released < 1% of the total radioactivity.
References
- Tchou, J. et al. (1994) Substrate specificity of Fpg protein. J. Biol. Chem., 269, 15318-15324.
- Hatahet, Z. et al. (1994) New substrates for old enzymes. J. Biol. Chem., 269, 18814-18820.
- Boiteux, S., O'Connor, T. and Laval, J. (1987) Formamidopyrimidine-DNA glycosylase of Escherichia coli: cloning and sequencing of the fpg structural gene and overproduction of the protein. EMBO J., 5, 3177-3183.
- 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.
- Czene, S. and Harms-Ringdahl, M. (1995) Detection of single strand breaks and formamidopyrimidine-DNA glycosylase-sensitive sites in DNA of cultured human fibroblasts. Mutat. Res., 336, 235-242.
Reagents Sold Separately
NEBuffer 1
BSA
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