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DNA Modifying Enzymes and Cloning >
DNA Repair Proteins >
Endonuclease IV |
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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 IV can act on a variety of oxidative damage in DNA (1). The enzyme is apurinic/apyrimidinic (AP) endonuclease that will hydrolyse intact AP sites in DNA. AP sites are cleaved at the first phosphodiester bond that is 5´ to the lesion leaving a hydroxyl group at the 3´ terminus and a deoxyribose 5´-phosphate at the 5´ terminus. The enzyme also has a 3´ -diesterease activity and can release phosphoglycoaldehyde, intact deoxyribose 5-phosphate and phosphate from the 3´ end of DNA.
Source:
An E.coli strain which carries the cloned Endo IV gene.
Applications:- Single cell gel electrophoresis (Comet assay)(2,3,4)
- Alkaline elution (5)
- Alkaline unwinding (6)
Reagents Supplied:
NEBuffer 3 (10X)
Enzyme Properties
Heat Inactivation:
No
Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer 3
Incubate at
37°C.
1X NEBuffer 3:
50 mM Tris-HCl
100 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 AP site* in a total reaction volume of 10 µl in 1 hour at 37°C.
*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
0.15% Triton X-100
pH 7.4 @ 25°C
Storage Temperature:
-20°C
Notes
Usage notes:- Recommended Dilution for the Comet Assay: 1.104 to 1:105(2,3,4,7).
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Quality Assurance Statement:
Purified free of contaminating exonucleases and endonucleases.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of λDNA (HindIII digest)
and 100 units of Endonuclease IV 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 10 units of Endonuclease IV 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 100 units of Endonuclease IV 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.
References
- Levin, J. et al. (1988) J. Gen. Physiol., 33, 349-362.
- Singh, N. et al. (1961) Experimental Cell Reseach, 175, 184-191.
- Collins, A. etal. (1993) Carcinogenesis, 14, 1733-1735.
- Collins, A. et al. (1996) Environmental Health Perspectives, 104, 465-469.
- Pflaum, M. et al. (1998) Free Rad. Res., 29, 585-594.
- Hartwig, A. et al. (1996) Toxicology Letters, 88, 85-90.
- Marks, K., New Entgland Biolabs, Inc., unpublished observations.
Reagents Sold Separately
NEBuffer 3
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