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  • Fpg

    Description

    Fpg (formamidopyrimidine [fapy]-DNA glycosylase) (also known as 8-oxoguanine DNA glycosylase) 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 both 3´ and 5´ to the AP site thereby removing the AP site and leaving a 1 base gap. Some of the damaged bases recognized and removed by Fpg include 7, 8-dihydro-8-oxoguanine (8-oxoguanine), 8-oxoadenine, fapy-guanine, methy-fapy-guanine, fapy-adenine, aflatoxin B1-fapy-guanine, 5-hydroxy-cytosine and 5-hydroxy-uracil (1,2).

    Highlights

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

    Product Source

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

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    NEBuffer 1-2010X
    BSA-2010 mg/ml

    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 a total reaction volume of 10 μl in 1 hour at 37°C.

    Reaction Conditions

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

    1X NEBuffer 1:
    10 mM Bis-Tris-Propane-HCl
    10 mM MgCl2
    1 mM DTT
    pH 7 @ 25°C

    Dilution for Comet Assay

    Recommended dilution for the Comet Assay: 1:103 to 1:104 (4,5,6). For a protocol please visit: http://cometassay.com.

    Storage Temperature

    -20°C

    Storage Conditions

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

    Heat Inactivation

    60°C for 10 min

    Unit Assay Conditions

    1X NEBuffer 1 containing 10 pmol of fluorescently labeled oligonucleotide duplex, supplemented with 100 µg/ml BSA in a total reaction volume of 10 μl.

    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.
    • 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):
      The product is tested for non-specific nuclease degradation in a reaction containing a DNA substrate. After incubation for 16 hours there is no detectable degradation of the DNA substrate 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.

    References

    1. Tchou, J. et al. (1994). Substrate specificity of Fpg protein. J. Biol. Chem.. 269, 15318-15324.
    2. Hatahet, Z. et al. (1994). New substrates for old enzymes. J. Biol. Chem.. 269, 18814-18820.
    3. Boiteux, S., O'Connor, T. and Laval, J. (1987). Formamidopyrimidine-DNA glycosylase of <em>Escherichia coli</em>: cloning and sequencing of the <em>fpg</em> structural gene and overproduction of the protein. EMBO J.. 5, 18814-18820.
    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. Czene, S. and Harms-Ringdahl, M. (1995). Detection of single strand breaks and formamidopyrimidine-DNA glycosylase-sensitive sites in DNA of cultured human fibroblasts. Mutat. Res.. 336, 235-242.

    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.
    1. What is the activity of Fpg in the NEBuffers 1-4?
    2. Does Fpg cleave hydroxymethyl uracil?
    3. Does Fpg cut only one strand, or do they cause a double-strand break?
    4. What is the Molecular Weight of Fpg?
    5. What is the difference between using Fpg and hOOG1?
    6. Does the Fpg contain a tag?
    7. What buffer will work with both Endonuclease III and Fpg?
    8. What substrate is Fpg tested on?
    1. Comet Assay - Modified for Detection of Oxidized Bases Using the Repair Endonucleases Fpg, hOGG1 and Endonuclease III (Nth)

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