TopoisomeraseI/IIInVivoLinkKit(ICEBioassay)ProductDescription
TopoGENhasextensiveexperiencewithassaysfortopoisomeraseinhibitioninvivo. Theseexperimentsallowtheinvestigatortoascertainwhetheranovelagentisactiveagainstendogenoustopoinachromosomalsettinginnuclei. Animportantbenefittothisanalysisisthatonecanuseanytumorcellortissueinordertoestablishclinicalefficacyofatestdrugagainstaspecifictumorcellline. WeuseessentiallythesamebasicapproachforTopoIasforTopoII. Themethodsarebaseduponphysicallyseparatingthetopo/DNAadductsfromfreeDNAandusingantibodiestomeasureboundtopoIorII. Inthisanalysis,tissueculturecellsaretreatedwithatestcompoundalongwithnegativecontrols(nodrug)andpositivecontrols(withknowninhibitors). ThecellsaredrugtreatedandrapidlylysedwithSarkosylwhichtrapssomefractionoftheendogenoustopoonDNAinacovalentcleavagecomplex. Followingdetergentlysis,thelysateisdilutedtofullydissociatenon-covalentDNA/proteincomplexes. Covalenttopo/DNAcomplexesareresolvedonastepCsClgrADIent(ionicDNA/proteininteractionsarealsopreventedby5MCsCl). ThegradientresolvesDNA,chromatinaggregates,andprotein,respectively.Gradientsarecentrifugedovernightandfractionated. TheamountoftopocoincidentwiththeDNApeakisameasureofcovalentDNA/topocomplexes. TopoconcentrationintheDNApeakisdeterminedbyimunoblottingusingantibodytotopoIorIIasprobe. IntheabsenceofagentsthatstABIlizethecleavablecomplex(etoposide,camptothecin)onlylowlevelsoftopoarefoundintheDNApeak;thisisparticularlyobviouswithtopoIIsincethetypeIIenzymeisnottrappedbythismethodunlessaninhibitorisused. Incontrast,topoIistrappedtoalowextentevenintheabsenceofcamptothecin. TheratiooftopoattheDNAdensityandtheproteindensityreflectstherelativeefficiencyofstabilizationofthecleavablecomplexes.

KitContents:

-Camptothecin(suppliedlyophilizedwithTG1021Top1)
-Etoposide(suppliedlyophilizedwithTG1022Top2)
-Sarkosyl(20%)
-AntibodytoTopoisomeraseI(SuppliedwithTG1021Top1)
-AntibodytoTopoisomeraseII(170kDaform,suppliedwithTG1022Top2)
-CsClStockSolution
-DetailedInstructionManual

GeneralProcedures:
Anoutlineofthemethodisshownbelow. Cellstobetestedmaybeaparticularcellline,virusinfectedcellortumortissue. Cellsareincubatedunderconditionsthatfavorendogenoustopoactivity(definedasphysiologicalconditionsconducivetocellgrowth);thus,theendogenousenzymeisengagingtheDNAtemplateinaseriesofbreakingandresealingsteps. Concurrently,centralgeneticprocessessuchasDNAreplication,transcriptionandrepairareongoing. Thecellsarethenrapidlylysedwithadetergent(sarkosyl). Itisimportantthatlysisbecarriedoutwhilemaintainingthecellsat37°C;ifthecellsarecooledormanipulatedpriortolysis,thecleavagecomplexestendtore-ligateandyieldnegativeresults(seeTraskandMuller,1988). ThenextsteprequirespurificationofDNAawayfromfreeprotein;however,organicextractionsorproteinasedigestionsmustbeavoided. WeuseastepCsClgradientforthispurpose. ThedensitystepsaredesignedtoresolveDNAfromfreeproteinandthereisquantitativerecoveryofboth.CovalentcomplexescontainingtopoandDNAsedimenttothepositionofDNA. ItisknownthatcovalentlyboundproteincanshiftthedensityofDNAinCsClandthemagnitudeofthedensityshiftisproportionaltothetotalamountofprotein. Invitro,topoImayproduceadensityshiftofDNAunderconditionsofstoichiometricexcessofprotein(datanotshown);however,invivosignificantlylessproteiniscoupledtoDNA. Infact,DNAtopoisomerasedoesnotcauseanydensityshiftofgenomicDNAinthisanalysis(seebelow)fortworeasons. First,thenumberofboundtopomeculesperDNAmoleculeisverylowandinthesegradients,complexesbehavelikefreeDNA(vs.DNA/proteinadducts). Second,thegradientsareverysteepanditisnotpossIBLetoresolvesmalldensitydifferencesanyway. AfterCsClcentrifugation,thegradientsarefractionatedandtheamountofboundandfreetopoismeasuredbyWesternblottingusingslotordotblotsandantibodiesdevelopedbyTopoGEN. Theratioofbound/freeisadirectmeasureofthecleavablecomplexformationintheparticularcellsystem. Wehavefoundthattopoisomerasesaredifficult(ifnotimpossible)totrapascleavablecomplexeswithincellsintheabsenceofinhibitors;thus,anytestcompoundthatresultsindetectionoftopoIorIIintheDNApeak(seebelow)ismostlikelyatopo-activeagent.TheInVivoLink-KitthenallowstheinvestigatortoevaluateacompoundforactivityagainsttopoIandIIindifferenttissuesettingsorwithvirusinfectedcells. Finally,itispossibletocombinetheanalysisoftopoIandIIinthesameexperiment. Inthiscase,onecanevaluatetopoisomeraseIinhibitionusingtheantibodytotopoisomeraseIortopoisomeraseII. Somedrugsmayconceivablyactuponbothenzymes(TraskandMuller,1988).

TopoGEN拓扑异构酶分析试剂盒，药物筛选试剂盒，新的ICE分析试剂盒，核提取试剂盒和补给品。拓扑异构酶测定试剂盒人类拓扑异构酶I检测试剂盒人拓扑异构酶II检测试剂盒旋转酶测定试剂盒DNA嵌入剂/展开试剂盒大肠杆菌DNA促旋酶和DNA松弛检测试剂盒TopI-NucEx试剂盒TopII-NucEx试剂盒SDS-K +沉淀试剂盒（体外）SDS-K +沉淀试剂盒（体内）药物筛选试剂盒拓扑异构酶I药物筛选试剂盒拓扑异构酶II药物筛选试剂盒拓扑异构酶II药物筛选试剂盒（基于kDNA）大肠杆菌拓扑异构酶IV药物筛选试剂盒大肠杆菌DNA促旋酶药物筛选试剂盒拓扑异构酶I高负荷抑制试剂盒基于细胞的拓扑测定试剂盒拓扑异构酶体内连接试剂盒人拓扑异构酶ICE检测试剂盒套件补给拓扑异构酶I测定缓冲液20毫米ATP10X凝胶加载缓冲液20％Sarkosyl大肠杆菌DNA促旋酶测定缓冲液金黄色葡萄球菌促旋酶测定缓冲液蛋白酶K拓扑异构酶II稀释缓冲液拓扑异构酶IV测定缓冲液谷氨酸钾拓扑异构酶II检测缓冲液拓扑异构酶I稀释缓冲液