TopoisomeraseIAssayKitProductDescription
ThiskitcontainsreagentsforroutinedetectionoftypeItopoisomeraseactivity.DNAMarkers(included)allowunequivocaldetectionoftheenzyme.TheassayisspecificfortopoisomeraseI;(eukaryotictypeIItopoisomerasesrequireMgCl2andATP).ThestandardtopoIassaykitissuppliedwithEDTAcontainingreactionbuffer(minusMg++)inordertopreventnucleolyticnickingofthesupercoiledsubstrate(althoughwenotethatsomeeukaryotictypeIactivitiesarestimulatedbyMg++).Thisassaywillworkevenwithcrudeextracts.Adetailedmanualofinstruction(seelinkabove)isincludedthatdescribeshowtorunthegelstomaximallyresolvetopoisomersforanalysisofDNAunwinding.

 TopoisomeraseIAssayKitContents(representativefor100assaykitsize):

-SupercoiledpHOT-1DNA(25µgin100µlTEbuffer,or0.25µg/ul)
-RelaxedpHOT-1DNAmarker,relaxedbytopoI(50ulat0.05µg/mlingelloADIngbuffer)
-10XTopoIassaybuffer(300µl)
-5XStopbuffer/gelloadingdye(600µl)
-Detailedinstructionmanual
-Sampledataanddocumentationofallcontrols

ReviewsandCitations:

• DexheimerTS,PommierY:DNAcleavageassayfortheidentificationoftopoisomeraseIinhibitors. Nature2008,3(11):1736-1750.doi:10.1038/nprot.2008.174
• ZhangL,SullivanP,SuyamaJ,et.al:Epidermalgrowthfactor-inducedheparanasenucleolarlocalizationaugmentsDNAtopoisomeraseIactivityinbrainmetastaticbreastcancer. MolecularCancerResearch2010,8:278-290.doi:10.1158/1541-7786.MCR-09-0375

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