Product Class: Nuclease

APE 1

cloned at neb recombinant NEBuffer 4 incubation temp heat inactivation
Catalog #SizeConcentration
M0282S1,000 units10,000 units/ml
M0282L5,000 units10,000 units/ml

Description

Highlights

  • Isolated from a recombinant source
  • Supplied with 10X Reaction Buffer
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).


Product Source

An E. coli strain which carries the cloned human APE 1 gene.

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
NEBuffer 4-2010X

Advantages and Features

Applications

  • Single cell gel electrophoresis (Comet assay)
  • Alkaline elution
  • Alkaline unwinding
  • Modified nick translation

Properties and Usage

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.

Reaction Conditions

1X NEBuffer 4
Incubate at 37°C

1X NEBuffer 4:
50 mM Potassium Acetate
20 mM Tris-acetate
10 mM Magnesium Acetate
1 mM DTT
pH 7.9 @ 25°C

Storage Temperature

-20°C

Storage Conditions

10 mM Tris-HCl
50 mM NaCl
1 mM DTT
0.05 mM EDTA
200 μg/ml BSA
50% Glycerol
pH 8.0 @ 25°C

Heat Inactivation

65°C for 20 min

Unit Assay Conditions

1X NEBuffer 4 containing 20 pmol of fluorescently labled oligonucleotide duplex in a total reaction volume of 10 μl.

Heat Inactivated

Yes

Quality Control

Quality Control Assays

The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
  • Endonuclease Activity (Nicking):
    The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
  • Exonuclease Activity (Radioactivity Release):
    The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
  • Protein Purity (SDS-PAGE):
    The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest band in a sample of known dilution. The purity is determined by SDS-PAGE.

Supporting Documents

Material Safety Datasheets

The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.

Notes

  1. Recommended Dilution for the Comet Assay: 1:103. For a protocol please visit: http://cometassay.com

References

  1. Robson, C.N. and Hickson, D.I. (1991). Nucl. Acids Res.. 19, 5519-5523.
  2. Demple, B. et al. (1991). Proc. Natl. Acad. Sci. USA. 88, 11450-11454.
  3. Barzilay, G. et al. (1995). Nucl. Acids Res.. 23, 1544-1550.
  4. Barzilay, G. et al. (1995). Nature Struc. Biol.. 2, 451-468.
  5. Gorman, M.A. et al (1997). EMBO J.. 16, 6548-6558.
  6. Xanthoudakis, S. et al. (1992). EMBO J.. 11, 3323-3335.
  7. Walker, L.J. et al. (1993). Mol. Cell. Biol.. 13, 5370-5376.
  8. Vidal, A.E. (2001). EMBO J.. 20, 6530-6539.
  9. Wilson, D.M. III et al. (1995). J. Biol. Chem.. 270, 16002-16007.
  10. Flaherty, D.M. (2001). Am. J. Respir. Cell. Mol. Biol.. 25, 664-667.
  1. What is the activity of APE 1 in NEBuffers 1-4?
  2. What is the molecular weight of APE 1?
  3. Is APE 1 a tagged protein?
  4. What substrate is used to test APE 1 activity?