Human Topoisomerase I Assay Kit contains reagents for routine detection of Topoisomerase I activity. DNA markers (included) allow unequivocal detection of the enzyme. The assay is specific for top1 enzyme; (eukaryotic type II topoisomerases require MgCl2 and ATP). This assay is relatively simple and measures loss of supercoiled DNA or 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
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