TopoisomeraseIVDrugScreeningKitBackground
TheTopoisomeraseIVDrugScreeningKitcontainsreagentsnecessarytoidentifycompoundsthataffectenzymeactivity. NotethatthiskitdoesnotincludeE.colitopoisomeraseIVenyzme,whichissoldseparately. Thiskitwillallowdetectionoftwokindsoftopoisomeraseinhibitors:thosethatstimulateformationofcleavablecomplexesandthosethatantagonizetopoisomerase(topo)IVactionontheDNA. SincetopoIVisatypeIIenzyme,wemayrefertoitaseithertopoIVortopoII. AknowntopoIIpoisonisincludedasacontrol. Theassaysystemisbaseduponincubatingsupercoiledplasmid(pHOT1)withTopoIVandevaluatingtheformationofDNAcleavageproductswhichmaybenicked,opencircularDNAorlinearDNA. Theproductsarethenresolvedbyethidiumbromidegelelectrophoresisasdescribedinthekit.
TopoisomeraseIVDrugScreeningKitContents(suitablefor100assays):
-pHOT1DNA,supercoiled25ug
-MarkerDNA,LinearpHOT1
-IncompleteBufferAcontainsthefollowing:200mMHEPES-KOH[pH7.6],500mMpotassiumglutamate,50mMMagnesiumAcetate,50mMDithiothreitol,250ugBSA/ml(500ul)*
-ATPBufferBcontains100mMATP*
*Thesetwobuffersmustbemixedbeforeusetomakethefinal5xCompleteTopoIVAssayBuffer(A+B)asfollows:add0.1volumeofBufferBto1.0volumeBufferA.PrepareonlyasmuchoftheCompleteTopoIVAssayBuffer(A+B)asneededforeachdayorsetofexperiments.Notethatthisbuffershouldbekeptoniceandusedthesameday,thendiscarded.
-Ciprofloxacincontroldrug
-10%sodiumdodecylsulfate(SDS)
-10xgelloADIngbuffer
-ProteinaseK
-Detailedinstructionmanual
ReviewsandCitations
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