TopoisomeraseIIDrugScreeningKit(kDNAbased)ProductDescription
ThisKitwillunambiguoslyidentifycompoundsthattargettopoisomeraseIIaction(topoII).ThekitdetectstwokindsofTopoIIeffectors:thosethatinhibittheactivityoftheenzyme(CatalyticInhibitoryCompoundsorCICs)andthosethatstimulateformationofthecleavagecomplexes(InterfacialPoisonsorIFPs).CICsmayaffectenzymeatoneofmanylevels,suchasblockingaccesstoDNAsubstrateoralteringATPaseactionandenzymeturnover.IFPsareanothertypeofinhibitorthatblockstheresealingstepofthereactiononDNAsuchthatDSDNAbreakstendtoaccumulate.Plasmidbasedkits,suchasTG1009,areidealfordetectingIFP(poisonslikeVP16).ThiskDNAbasedDrugKitisoptimizedfordetectingtopoIIcatalyticinhibitors(CIC)typeofdrugs.Undernormalcircumstances,TopoIIentersintoabreakage/resealingcyclethatfavorstheresealedproduct(Fig1).Thecleavageintermediatehasanextremelyshortlifetimeandcannotbeidentified.AknownTopoIIpoison(etoposideorVP16)isincludedasacontrolIFPsinceapositivedrugisrequiredtoensurethattheassayisworkingproperlyandiscapableofresolvingunknownIFPmechaNISTicdrugs.TheDNAsubstrate(kDNA)includedinthisassayisidealbecauseitisaveryattractivesubstratefortopoIIcatalysis.ThiskDNAassaysystemisbaseduponevaluatingeithertheformationofDNAcleavageproducts(linearkDNA)forIFPs.ForCICdetection,theassaysimplymeasuresinhibitionofthedecatenaseactivityoftopoII.Inthiscase,thereisaclearlossofdecatenatedkDNAproducts.Theabovevideohasmoreinformation.

TopoisomeraseIIDrugScreeningKitContents(kDNA-based):

-kDNASubstrate
-DecatenatedandLinearizedkDNAMarkers
-10XTopoisomeraseIIassaybuffercomponents*

*ToimprovestABIlity,the10xTopoIIAssaybufferisprovidedastwocomponents: BufferA(noATP)andBufferB(ATP).TheComplete10xAssaybufferismadefresheachtimebycombiningBufferAandB.

-VP-16(etoposide)
-Sodiumdodecylsulfate(10%)
-10XGelloADIngbuffer(bromophenolblue,glycerol)
-ProteinaseK
-Detailedinstructionmanual

Note:TopoIIenzymeissoldseparately.