Product Class: Other

Endonuclease VIII
Gray property icon for NEBU Gray property icon for cloned at NEB recombinant Gray property icon for incubation temperature 37° 75 Heat

Product Introduction

Diagram of mechanism


  • Bifunctional DNA glycosylase with DNA N-glycosylase and AP lyase activities
  • The N-glycosylase activity releases damaged pyrimidines, including thymine glycol and uracil glycol. The AP lyase activity cleaves DNA phosphodiester backbone at AP sites via β and δ-elimination, creating a 1 nucleotide DNA gap with 5' and 3' phosphate termini
Bioz Badge Exists : True
Catalog # Size Concentration
M0299S 1000 units 10000 units/ml
M0299L 5000 units 10000 units/ml

Product Information

Description

Endonuclease VIII from E. coli acts as both an N-glycosylase and an AP-lyase. The N-glycosylase activity releases damaged pyrimidines from double-stranded DNA, generating an apurinic (AP site). The AP-lyase activity cleaves 3´ and 5´ to the AP site leaving a 5´ phosphate and a 3´ phosphate. Damaged bases recognized and removed by Endonuclease VIII include urea, 5, 6- dihydroxythymine, thymine glycol, 5-hydroxy-5- methylhydantoin, uracil glycol, 6-hydroxy-5, 6-dihydrothymine and methyltartronylurea (1,2). While Endonuclease VIII is similar to Endonuclease III, Endonuclease VIII has β and δ lyase activity while Endonuclease III has only β lyase activity.

Product Source

An E. coli strain which carries the cloned nei gene.
This product is related to the following categories:
DNA Repair Enzymes and Structure-specific Endonucleases,

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • NEB # Stored at (°C)
    M0299S -20
    Component Name Component # Stored at (°C) Amount Concentration
    Endonuclease VIII M0299SVIAL -20 1 x 0.1 ml 10,000 units/ml
    Endonuclease VIII Reaction Buffer B0299SVIAL -20 1 x 1.5 ml 10 X
  • NEB # Stored at (°C)
    M0299L -20
    Component Name Component # Stored at (°C) Amount Concentration
    Endonuclease VIII M0299LVIAL -20 1 x 0.5 ml 10,000 units/ml
    Endonuclease VIII Reaction Buffer B0299SVIAL -20 1 x 1.5 ml 10 X

Properties & Usage

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 VIII 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.

Reaction Conditions

1X Endonuclease VIII Reaction Buffer
Incubate at 37°C

1X Endonuclease VIII Reaction Buffer
10 mM Tris-HCl
75 mM NaCl
1 mM EDTA
(pH 8 @ 25°C)

Storage Buffer

10 mM Tris-HCl
250 mM NaCl
0.1 mM EDTA
50% Glycerol
pH 8 @ 25°C

Heat Inactivation

75°C for 10 minutes

Unit Assay Conditions

1X Endonclease VIII Reaction Buffer containing 10 pmol of fluorescently labeled oligonucleotide duplex in a total reaction volume of 10 μl.

Application Features

  • Single cell gel electrophoresis (Comet assay) (3,4,5)
  • Alkaline elution (6)
  • Alkaline unwinding (7)

Product Notes

  1. Recommended Dilution for Comet Assay: 1:104 to 1:105 (3,4,5,8). For a protocol please visit: http://cometassay.com
  2. Endonuclease VIII has full activity in CutSmart Buffer (NEB #B7204). Endo VIII has a 75% activity in NEBuffer 1 (NEB #B7001), 75% activity in NEBuffer 4 (NEB #B7004), 50% activity in NEBuffer 2 (NEB #B7002). The use of Endonuclease VIII in NEBuffer 3 (NEB #B7003) is not recommended. 

References

  1. Dizdaroglu, M., Laval, J. and Boiteux, S. (1993). Biochemistry . 32, 12105-12111.
  2. Hatahet, Z. et al. (1994). J. Biol. Chem.. 269, 18814-18820.
  3. Singh, N. et al. (1988). Exp. Cell. Res. . 175, 184-191.
  4. Collins, A. et al. (1993). Carcinogenesis . 14, 1733-1735.
  5. Collins, A. et al. (1996). Environ. Health Perspect . 104, 465-469.
  6. Pflaum, M. et al. (1998). Free Rad. Res. . 29, 585-594.
  7. Hartwig, A. et al. (1996). Toxicol. Lett.. 88, 85-90.
  8. Marks, K. unpublished observation.

Tools & Resources

Selection Charts

FAQs & Troubleshooting

FAQs

  1. What is the activity of Endonuclease VIII in NEBUffers?
  2. Does Endonuclease VIII cleave RNA?
  3. Does treatment of DNA with Endonuclease VIII leave a 5' phosphate?
  4. What damaged bases does Endonuclease VIII recognize?
  5. Will Endonuclease VIII work in rCutSmart buffer?

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

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This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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