TopoisomeraseIDrugScreeningKitProductDescription
TheTopoIDrugKitintroducestransientnicksinDNAatspecificsites. DetectionofthesetransientDNAnicksrequirestrappingtheenzymeonDNAinanickedintermediatecomplexusingproteindenaturants.TheresultingcovalentDNA/topoIcomplexescontainnickedopencircularDNAwhichcanbedetectedbyagarosegelelectrophoresis(withethidiumbromide). Trappingnickedintermediatesisrelativelyinefficient;however,inhibitors,suchasthenaturalproductcamptothecin,stABIlizetheintermediateandleadtoanincreaseinthenickedDNAproduct. ThisformsthebasisforamechaNISTicdrugscreeningassaydesignedtoallowdetectionofagentsthataffecttopoisomeraseIbystabilizingthecleavedintermediatecomplex. TheTopoGENTopoIDrugScreeningKitisdesignedtoallowtheinvestigatortoquicklyidentifynovelinhibitorsoftopoIenzyme(soldseparately). ThekitwillallowdetectionofnovelcompoundsthateitherstabilizethenickedintermediateorotherwiseinhibitcatalyticactivityoftopoisomeraseI.

TopoisomeraseIDrugScreeningKitContents(representativefor100assaykitsize):

-SupercoiledpHOT-1DNA,25µg
-RelaxedandnickedplasmidDNAMarkers
-10XTopoisomeraseIassay/cleavagebuffer,300ul
-Sodiumdodecylsulfate(SDS)terminationbuffer(10%)
-10XgelloADIngdye
-Controlinhibitor(Camptothecin;lyophilized)
-ProteinaseK
-Detailedinstructionmanual

Note:TopoisomeraseIenzymesoldseparately.

TheTopoisomeraseIDrugScreeningKitisdesignedtoworkwithhumantopoIavailablefromTopoGEN.Sincecleavageassaysrequiremoreenzymethancatalyticassays,TopoGENsellstheenzymeinlargerlotswhichtakesthisdifferenceintoaccount.

UseoftheTopoisomeraseIDrugKit
Reactionsindicatedaboveeachlanewerecarriedoutusing5unitsofTopoGENtypeItopoisomerase. Thereactionswereincubatedfor30minat37CandstoppedwithSDS. AfterdigestionwithproteinaseK(50ug/ml,60minat37C),sampleswereloadedontoa1%agarosegel. Undertheseconditions,withexcesstopoIandcamptothecin,significantconversionofsupercoiled(formI)DNAtoopencircular(OCDNA)wasobserved. Itisimportanttorecognizethatcleavageassayswillconsumemoreenzymethancatalytic(relaxationtype)assays. Thisisbecauseeachcleavageeventconsumesoneenzymemolecule;i.e.,thereactionrequiresstoichiometicamountsoftopoI.

TopoGEN人类拓扑异构酶IIα单克隆抗体SKU尺寸价钱数量TG2010-150伙$ 285.00                  1个                  2                  3                  4                  5                  6                  7                  8                  9                  11                   添加到购物车人类拓扑异构酶IIα单克隆抗体：免疫印迹和免疫组化试剂制备了针对源自p170 kDa人Topo IIa C端的寡肽的单克隆抗体。然后在含有相同肽的柱子上亲和纯化抗体。亲和纯化的抗体可用于探测Western印迹，前提是包括对照以清楚地标记170 kDa topo II的位置。原因是在常规提取和操作过程中人topo II很容易降解或被蛋白水解。结果，可以检测子带。一些实验室已经报道了SDS-PAGE模式的异质性（Shelton等，1983; Halligan等，1985; Muller等，1988），最近还报道了粟酒裂殖酵母拓扑异构酶II的蛋白水解产物（Shiozaki和Yanagida，1991年）。因此，如果存在广泛的蛋白水解，抗体可能看起来是非特异性的。TG2011-3），其对应于完整的170kDa拓扑异构酶II。报道了编码汉拓扑异构酶II的第二个基因（拓扑II-β或180kDa形式）（Chung等，1989； Tan等，1992）。该抗体与topo II的170 kDa亚型特异性反应。单克隆抗体单位定义：抗体浓度以蛋白质印迹单位为单位；一个单位对应于制备常规蛋白质印迹稀释探针所需的抗体的1：1000稀释度。因此，10个单位的抗体将制成10 ml稀释的探针（直接添加到单个印迹中）。