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DNA Modifying Enzymes and Cloning >
DNA Repair Proteins >
Endonuclease III (Nth) |
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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:
Endonuclease III (Nth) protein from E. coli acts as both N-glycosylase and a AP-lyase. The N-glycosylase activity releases damaged pyrimidines from double-stranded DNA, generating a basic (AP site). The AP-lyase activity of the enzyme cleaves 3´ to the AP site leaving a 5´ phosphate and a 3´ ring opened sugar. Some of the damaged bases recognized and removed by Endouclease III include urea, 5, 6 dihydroxythymine, thymine glycol, 5-hydroxy-5 methylhydanton, uracil glycol, 6-hydroxy-5, 6-dihdrothimine and methyltartronylurea (1,2).
Source:
An E. coli strain which carries the cloned nth gene from Escherichia coli.
Applications:- Single cell gel electrophoresis (Comet assay) (3,4,5)
- Alkaline elution (6)
- Alkaline unwinding (7)
Reagents Supplied:
Endonuclease III (Nth) Reaction Buffer (10X)
Enzyme Properties
Heat Inactivation:
No
Reaction & Storage Conditions
Reaction Conditions:
1X Endonuclease III (Nth) Reaction Buffer
Incubate at
37°C.
1X Endonuclease III (Nth) Reaction Buffer:
20 mM Tris-HCl
1 mM Dithiothreitol
1 mM EDTA
pH 8.0 @ 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 AP site* in a total reaction volume of 10 µl in 1 hour at 37°C in 1X Endonuclease III Reaction Buffer containing 10 pmol of fluorescently labeled oligonucleotide duplex.
* An AP site is created by treating 10 pmol of a 34 mer oligonucleotide duplex containing a single uracil residue with 1 unit of Uracil-DNA Glycosylase (UDG) for 2 minutes at 37°C.
Concentration:
10,000 units/ml
Storage Conditions:
10 mM Tris-HCl
250 mM NaCl
1 mM Dithiothreitol
0.1 mM EDTA
200 µg/ml BSA
50% Glycerol
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.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 1,000 units of Endonuclease III (Nth) with 1 μg of
ΦX174 RF I DNA for 4 hours at 37ºC resulted
in < 10% conversion to RFII as determined by agarose gel electrophoresis.
RNase Assay Radioactive:
Incubation of 600 units of enzyme with 20 nmol [3H]poly(A).poly(dT) hybrid polymer for
1 hour at 37ºC in a 50 μl reaction released < 1
nmol adenosine-5'-monophosphate.
Double-stranded Exonuclease Assay::
Incubation of 1000 units of enzyme with 1 µg sonicated [3H] DNA (105 cpm/µg) for 4 hours at 37°C in 50 µl reaction buffer released < 0.1% radioactivity.
SS DNA Exonuclease Activity:
Incubation of 1 units of enzyme with 1 μg sonicated and denatured [3H]-DNA (105 cpm/μg) for 4 hours at 37°C in 50 μl reaction buffer released < 10% radioactivity.
References
- Dizdaroglu, M., Laval, J. and Boiteux, S. (1993) Biochemistry, 32, 12105-12111.
- Hatahet, Z. et al. (1994) J. Biol. Chem., 269, 18814-18820.
- Singh, N. et al. (1988) Exp. Cell Res., 175, 184-191.
- Collins, A. et al. (1993) Carcinogenesis, 14, 1733-1735.
- Collins, A. et al. (1996) Environ. Health Perspect., 104, 465-469.
- Pflaum, M. et al. (1998) Free Rad. Res., 29, 585-594.
- Hartwig, A. et al. (1996) Toxicol. Lett., 88, 85-90.
Reagents Sold Separately
Endonuclease III (Nth) Reaction Buffer
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