Thermo Fisher Scientific Endonuclease IV, E.coli User guide

Brand
Thermo Fisher Scientific
Model
Endonuclease IV, E.coli
Type
User guide
PRODUCT INFORMATION
Endonuclease IV,
E.coli (Endo IV)
Pub. No. MAN0012008
Rev. Date 29 November 2016 (Rev. B.00)
#_
Lot: _ Expiry Date: _
Store at -20 °C
Components
#EN0591
Endonuclease IV,
E.coli (Endo IV)
2 U/µL
100 U
10X Reaction Buffer
1 mL
www.thermofisher.com
For Research Use Only. Not for use in diagnostic procedures.
Description
Endonuclease IV (Endo IV) recognizes
apurinic/apyrimidinic (AP) sites of dsDNA and cleaves the
phosphodiester bond 5' to the lesion generating a
hydroxyl group at the 3'-terminus.
The enzyme can also act as a 3'-diesterase that is able
to release 3'-phosphoglycolate or 3'-phosphate from
damaged ends of dsDNA (1).
Endo IV possesses also a 3'→5' exonuclease activity. Its
progression on substrates is sensitive to ionic strength,
metal ions, EDTA, and reducing conditions. Substrates
with 3'-recessed ends are preferred substrates for 3'→5'
exonuclease activity (2).
The enzyme has no requirement for Mg2+ but is more
active in the presence of Mg2+.
Applications
Studies of DNA damage and repair (3, 4, 5).
Single cell electrophoresis (comet assay) (6).
Antitumor drug research (4).
DNA structure research (5, 7).
SNP analysis (8).
Source
E.coli cells with a cloned nfo gene.
Definition of Activity Unit
One unit of the enzyme relaxes 1 µg of partially
depurinated, supercoiled plasmid DNA in 30 min at 37 °C.
Enzyme activity is assayed in the following mixture:
50 mM Tris-acetate (pH 7.5), 50 mM KCl, 1 mM EDTA,
0.05% (v/v) Triton X-100 and 2 µg of partially depurinated
pUC19 DNA.
Storage Buffer
Enzyme is supplied in: 50 mM Tris-HCl (pH 7.5),
100 mM NaCl, 1 mM DTT, 0.1% (v/v) Triton X-100 and
50% (v/v) glycerol.
10X Reaction Buffer
500 mM Tris-acetate (pH 7.5), 500 mM KCl,
10 mM EDTA, 0.5% (v/v) Triton X-100.
Inhibition and Inactivation
Inhibitors: the enzyme is fairly resistant to EDTA during
the reaction, but becomes sensitive to even submillimolar
quantities of chelators when no DNA substrate is
present.
Inactivated by heating at 80 °C for 15 min.
CERTIFICATE OF ANALYSIS
Endodeoxyribonuclease Assay
No detectable degradation was observed after incubation
of supercoiled plasmid DNA with Endonuclease IV.
Ribonuclease Assay
No detectable degradation was observed after incubation
of [3H]-RNA with Endonuclease IV.
Quality authorized by: Jurgita Zilinskiene
(continued on reverse page)
References
1. Levin, J.D., Homogeneous Escherichia coli endonuclease
IV, J. Biol. Chem., 263, 8066-8071, 1988.
2. Kerins, S.M., et al., Characterization of an endonuclease IV
3’-5’ exonuclease activity, J. Biol Chem. Jan. 31,278(5),
3048-3054, 2003.
3. Demple, B., Harrison, L., Repair of oxidative damage to
DNA: Enzymology and biology, Annu. Rev. Biochem., 63,
915-948, 1994.
4. Levin, J.D., Demple, B., In vitro detection of endonuclease
IV – specific DNA damage formed by bleomicin in vivo,
Nucleic Acids Res., 24, 885-889, 1996.
5. Patro, J.N., et al., Probing the configurations of
formamidopyrimidine lesions Fapy.dA and Fapy.dG in DNA
using endonuclease IV, Biochemistry, 43, 13397-13403,
2004.
6. Collins, A.R., The comet assay for DNA damage and repair,
Molec. Biotechnol., 26, 249-261, 2004.
7. Hosfield, D.J., et al., Structure of the DNA repair enzyme
endonuclease IV and its DNA complex: double-nucleotide
flipping at abasic sites and three-metal-ion catalysis, Cell,
98, 397-408, 1999.
8. Kutyavin I.V., et al., A novel endonuclease IV post-PCR
genotyping system, Nucleic Acids Res., 29, 1-9, 2006
LIMITED USE LABEL LICENSE: Internal Research and Development Use
Only.
The purchase of this product conveys to the buyer the limited, non-exclusive, non-
transferable right (without the right to resell, repackage, or further sublicense) to use
this product for internal research and development purposes. No other license is
granted to the buyer whether expressly, by implication, by estoppel or otherwise. In
particular, the purchase of the product does not include or carry any right or license
to use, develop, or otherwise exploit this product commercially and no rights are
conveyed to the buyer to use the product or components of the product for purposes
including but not limited to provision of services to a third party, generation of
commercial databases or clinical diagnostics. This product is sold pursuant to
authorization from Thermo Fisher Scientific and Thermo Fisher Scientific reserves all
other rights. For information on purchasing a license for uses other than internal
research and development purposes, please contact [email protected] or
Out Licensing, Life Technologies Inc., 5781 Van Allen Way, Carlsbad, California
92008.
PRODUCT USE LIMITATION
This product is developed, designed and sold exclusively for research purposes and
in vitro use only. The product was not tested for use in diagnostics or for drug
development, nor is it suitable for administration to humans or animals.
Please refer to www.thermofisher.com for Material Safety Data Sheet of the product.
© 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the
property of Thermo Fisher Scientific Inc. and its subsidiaries.
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Thermo Fisher Scientific Endonuclease IV, E.coli User guide

Brand
Thermo Fisher Scientific
Model
Endonuclease IV, E.coli
Type
User guide
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