hOGG1

This product has been discontinued. We recommend Fpg as alternative.
  • This product was discontinued on 06/15/2017
recombinant unique buffer incubation temp heat inactivation bsa
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Categories:
Discontinued Products
Applications:
DNA Nicking,
DNA Repair

Description

hOGG1 (α isoform) is an 8-oxoguanine DNA glycosylase which acts both as a N-glycosylase and an AP-lyase. The N-glycosylase activity releases damaged purines from double stranded DNA, generating an apurinic (AP) site. The AP-lyase activity cleaves 3´ to the AP site leaving a 5´ phosphate and a 3´-phospho-α, β-unsaturated aldehyde.

Some of the damaged bases recognized and removed by hOGG1 include 7, 8-dihydro-8-oxoguanine (8-oxoguanine) when base paired with cytosine, 8-oxoadenine when base paired with cytosine, foramidopyrimidine (fapy)-guanine and methy-fapy-guanine (1,2).


Highlights

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

Product Source

An E. coli strain that carries the cloned human ogg1 gene (3).

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
NEBuffer™ 2-2010X
Purified BSA-2010 mg/ml

Advantages and Features

Applications

  • Single cell gel electrophoresis (Comet assay) (4,5,6)
  • Alkaline elution (7)
  • Alkaline unwinding (8)

Properties and 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 8-oxoguanine base paired with a cytosine in 10 µl of 1X NEBuffer 2 containing 10 pmol of substrate, supplemented with 100 µg/ml BSA in 1 hour at 37°C.

Reaction Conditions

1X NEBuffer 2
Supplement with 100 μg/ml Purified BSA
Incubate at 37°C

1X NEBuffer™ 2:
50 mM NaCl
10 mM Tris-HCl
10 mM MgCl2
1 mM DTT
pH 7.9 @ 25°C

Dilution for Comet Assay

1:102 to 1:103 (4,5,6,9). For a protocol please visit: http://cometassay.com

Storage Temperature

-20°C

Storage Conditions

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

Heat Inactivation

65°C for 15 min

Unit Assay Conditions

1X NEBuffer 2 containing 10 pmol of fluorescently labeled oligonucleotide duplex, supplemented with 100 µg/ml BSA.

References

  1. Bjoras, M. et al. (1997). Opposite base-dependent reactions of a human base excision repair enzyme on DNA containing 7, 8-dihydro-8-oxoguanine and abasic sites. EMBO J. . 16, 6314-6322.
  2. Boiteux, S. and Radicella, J. (1999). Base excision repair of 8-hydroxyguanine protects DNA from endogenous oxidative stress. Biochimie . 81, 59-67.
  3. Radicella, J., Dherin, C., Desmze, C., Fox, M. and Boiteux, S. (1997). Cloning and characterization of hOGG1, a human homolog of the OGG1 gene of Saccharomyces cerevisiae. Proc. Natl. Acad. Sci USA. 94, 8018-8015.
  4. Singh, N., McCoy, M., Tice, R. and Schneider, L. (1988). A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell. Res. 175, 184-191.
  5. Collins, A., Duthie, S. and Dobson, V. (1993). Direct enzymatic detection of endogenous oxidative base damage in human lymphocyte DNA. Carcinogenesis . 14, 1733-1735.
  6. Collins, A., Dusinska, M., Gedik, C. and Stetina, R. (1996). Oxidative damage to DNA: do we have a reliable biomarker?. Environ. Health Perspect. . 104, 465-469.
  7. Pflaum, M., Will, O., Mahler, H.-C. and Epe, B. (1998). DNA oxidation products determined with repair endonucleases in mammalian cells: types, basal levels and influence of cell proliferation. Free Rad. Res. 29, 585-594.
  8. Hartwig, A., Dally, H. and Schlepegrell, R. (1996). Sensitive analysis of oxidative DNA damage in mammalian cells: use of the bacterial Fpg protein in combination with alkaline unwinding. Toxicol. Lett. . 88, 85-90.
  9. Gutherie, E. unpublished observation.

FAQs

  1. What is the difference between hOGG1 and FPG?
  2. What is the activity of hOGG1 in the NEBuffers?
  3. What substrate is used to test hOGG1?
  4. What is the molecular weight of hOGG1?
  5. Is hOGG1 a tagged protein?

Protocols

  1. Comet Assay - Modified for Detection of Oxidized Bases Using the Repair Endonucleases Fpg, hOGG1 and Endonuclease III (Nth)

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.

Selection Charts

Usage Guidelines & Tips

Interactive Tools

Citations

  • Karbaschi, Mahsa., Macip, Salvador., Mistry, Vilas., Abbas, Hussein H.K., Delinassios, George J., Evans, Mark D., Young, Antony R., Cooke, Marcus S. (2015). Rescue of cells from apoptosis increases DNA repair in UVB exposed cells: implications for the DNA damage response Toxicol. Res.. 4, 725-738. DOI: doiL 10.1039/c4tx00197d

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. Further information regarding NEB product quality can be found here.
  • 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.
  • Non-Specific DNase Activity (16 hour, DNA):
    The DNA is tested in a reaction under standard reaction conditions. After incubation for 16 hours there is no detectable degradation of the DNA as determined by agarose gel electrophoresis.
  • 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.

Safety Data Sheet

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

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.