Human Topoisomerase I Assay Kit contains all the necessary reagents for routine detection of Topoisomerase I activity. The included DNA markers allow unequivocal detection of the enzyme. The assay is specific for topoisomerase I enzyme; (eukaryotic type II topoisomerases require MgCl2 and ATP). Supplied in kit form, this assay is relatively simple and measures loss of supercoiled DNA, or the appearance of relaxed DNA products.
The standard topo I assay kit is supplied with EDTA containing reaction buffer (minus Mg++) in order to prevent nucleolytic nicking of the supercoiled substrate (although we note that some eukaryotic type I activities are stimulated by Mg++). DNA Markers are included to allow unequivocal detection of activity.
The Human Topo I Assay Kit will work with crude extracts as well. A detailed manual of instruction is included that describes how to run the gels to maximally resolve topoisomers for analysis of DNA unwinding.
Topoisomerase I Assay Kit Contents (representative for 100 assay kit size)
-100 Units Human Top1 Enzyme (available with catalog number TG1015-1A only)
-Supercoiled pHOT-1 DNA (25 µg in 100 µl TE buffer, or 0.25 µg/ul)
-Relaxed pHOT-1 DNA marker, relaxed by topo I (50 ul at 0.05 µg/ml in gel loading buffer)
-10X Topo I assay buffer (300 µl)
-5X Stop buffer/gel loading dye (600 µl)
–Detailed instruction manual
-Sample data and documentation of all controls
This kit is shipped at ambient temperature or on dry ice if purchased with enzyme. Store enzyme at -70° C. All other components may be stored at 4°C. Avoid frequent freeze/thaw cycles with the plasmid as this may contribute to DNA breakage.
Human Topoisomerase I Assay Kit Protocol
Material Safety Data Sheet
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Reviews and Citations
- Dexheimer TS, Pommier Y: DNA cleavage assay for the identification of topoisomerase I inhibitors. Nature 2008, 3(11): 1736-1750. doi:10.1038/nprot.2008.174
- Zhang L, Sullivan P, Suyama J, et. al: Epidermal growth factor-induced heparanase nucleolar localization augments DNA topoisomerase I activity in brain metastatic breast cancer. Molecular Cancer Research 2010, 8: 278-290. doi: 10.1158/1541-7786.MCR-09-0375