| Source: |
E. coli Fpg is purified from E. coli containing a recombinant plasmid harboring the E. coli Fpg+ gene. |
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| Unit Definition: |
One unit is the amount of enzyme
required to cleave 1 pmole of a 32P-labeled oligonucleotide
probe containing 8-oxoguanine within an oligonucleotide duplex
in one hour at 37°C.
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| Assay Conditions: |
4 pmoles of 32P-8-oxo-dG-oligonucleotide
probe, 4 pmoles of complementary oligonucleotide, 1X REC™
Buffer 10 (10 mM HEPES-KOH (pH 7.4), 100 mM KCl, 10 mM EDTA,
and 0.1 mg/ml BSA) for 1 hour at 37°C in a reaction volume
of 20 µl.
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| Applications: |
CometAssayTM/FLARETM
Supercoiled relaxation assays
DNA fragmentation analysis
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| Storage: |
Freeze in working aliquots at -80°C to avoid repeated freeze-thawing. Fpg is supplied in 20 mM Tris-Cl (pH 7.8), 100 mM NaCl, 1 mM EDTA, 1 mM DTT, and 50% glycerol. |
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| Specificity: |
E. coli Fpg catalyzes the excision of the following forms of DNA damage:
Open ring forms of 7-methylguanine, including 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine and 4,6-diamino-5-formamidopyrimidine, a lethal lesion; 8-oxoguanine, a highly mutagenic lesion and probably the most important biological substrate of Fpg; 5-hydroxycytosine; 5-hydroxyuracil; Aflatoxin-bound imidazole-ring-opened guanine; Imidazole ring opened N-2-aminofluorene-C8-guanine.
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