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DNA Modifying Enzymes and Cloning >
DNA Repair Proteins >
APE 1 |
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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:
Human apurinic/apyrimidinic (AP) endonuclease, APE 1, also known as HAP 1 or Ref-1, shares homology with Escherichia coli exonuclease III protein. APE 1 cleaves the phosphodiester backbone immediately 5´ to an AP site, via hydrolytic mechanism, to generate a single-strand DNA break leaving a 3´-hydroxyl and 5´ -deoxyribose phosphate terminus. Besides AP endonuclease activity, APE 1 has also been reported to have weak DNA 3´ -diesterase, 3´ to 5´ exonuclease and RNase H activities (3-5).
In addition to DNA repair activity, APE 1 is also capable of regulating the DNA binding activity of many transcription factors in vitro by a redox mechanism (Ref-1). As part of this process, APE 1 has been shown to stimulate the DNA binding activity of Fos-Jun heterodimers, Jun-Jun homodimers and Hela cell AP-1 proteins as well as that of several other transcription factors including NF-kB, Myb and members of the ATF/CREB family (7-9).
Source:
An E. coli strain which carries the cloned human APE 1 gene
Applications:- Single cell gel electrophoresis (Comet assay)
- Alkaline elution
- Alkaline unwinding
- Modified nick translation
Reagents Supplied:
NEBuffer 4 (10X)
Enzyme Properties
Heat Inactivation:
No
Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer 4
Incubate at
37°C.
1X NEBuffer 4:
20 mM Tris-acetate
50 mM potassium acetate
10 mM Magnesium Acetate
1 mM Dithiothreitol
pH 7.9 @ 25°C
Unit Definition:
One unit is defined as the amount of enzyme required to cleave 20 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 20 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
50 mM NaCl
1 mM Dithiothreitol
0.05 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.
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 100 units of APE 1 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.
Double-stranded Exonuclease Assay:
Incubation of 50 units of enzyme with 1 µg sonicated [3H] DNA (2 x 105 cpm/µg) for 4 hours at 37°C in 50 µl reaction buffer released < 0.23% radioactivity.
SS DNA Exonuclease Activity:
Incubation of 50 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 < 0.30% radioactivity.
References
- Robson, C.N. and Hickson, D.I. (1991) Nucl. Acids Res., 19, 5519-5523.
- Vidal, A.E. (2001) EMBO J., 20, 6530-6539.
- Demple, B. et al. (1991) Proc. Natl. Acad. Sci. USA, 88, 11450-11454.
- Barzilay, G. et al. (1995) Nucl. Acids Res., 23, 1544-1550.
- Barzilay, G. et al. (1995) Nature Struc. Biol., 2, 451-468.
- Wilson, D.M. III et al. (1995) J. Biol. Chem., 270, 16002-16007.
- Gorman, M.A. et al (1997) EMBO J., 16, 6548-6558.
- Xanthoudakis, S. et al. (1992) EMBO J., 11, 3323-3335.
- Walker, L.J. et al. (1993) Mol. Cell. Biol., 13, 5370-5376.
- Flaherty, D.M. (2001) Am. J. Respir. Cell. Mol. Biol., 25, 664-667.
Reagents Sold Separately
NEBuffer 4
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