These HR/NHEJ kits are cell based, meaning they have the HR or NHEJ GFP reporters pre-installed. A single plasmid (I-Sce1) is transfected to activate repair. Please see this table for a summary and comparison/application of each kit.
Transfection-based HR and NHEJ kits use transient transfections to make a pool of cells containing the DNA reporters to allow the investigator to interrogate the 2 major DNA repair pathways. The advantages are:
It uses GFP readout to measure the efficiency of repair.
There is zero background when repair is blocked or inhibited
The reporters for HR or NHEJ can be installed in virtually any cell line that can be transiently transfect
It is rapid and robust
It is ideal for drug screens to find novel inhibitors of HR/NHEJ
The kits are highly specific and can only report HR or NHEJ
Readout GFP can be done by FACS or Imaging
Entire pathway can be followed by live imaging in viable cells
Ideal to track repair kinetics at the single cell level
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Human Topoisomerase I Assay Kit
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Human Topoisomerase I Assay Kit
Starting from $320.00
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).
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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.
Are you interested in learning about how we can help your lab with your compound screening research? TopoGEN offers powerful and tractable services to identify Topoisomerase-targeting drugs In Vitro. Learn more about these services here.
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
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