Description
CatalogueNumber	S7810
BrandFamily	Chemicon®
TradeName	CpGWiz Chemicon
Description	CpGWIZ®RB1-MethylationspecificPCRassay
Overview	MSP,performedusingtheCpGenome™DNAModificationKitandtheCpGWIZ®RB1AmplificationKit,permitssensitivedetectionofalteredDNA.BecausethisisaPCR-basedassay,itisextremelysensitive,facilitatingthedetectionoflownumbersofmethylatedallelesandthestudyofsamplescontainingsmallamountsofDNA.MSPalsoallowsexaminationofallCpGsites,notjustthosewithinsequencesrecognizedbymethylationsensitiverestrictionenzymes.Increasingthenumberofsuchsiteswhichcanbeassessedallowsrapid,finemappingofmethylationpatternsthroughoutCpGregions.Inaddition,thebisulfitemodificationisideallysuitedforanalysisofCpGislandssinceitconvertsthemajorityofcytosinestouracils,makingaregionofthegenomewhichisCG-richlessdifficulttoamplifybyPCR. Methylation-specificPCR(MSP)employsaninitialbisulfitereactiontomodifytheDNA,followedbya"hotstart"PCRamplificationwithspecificprimersdesignedtodistinguishmethylatedDNAfromunmethylatedDNA.AsshowninFigure1,inthebisulfitereaction,allunmethylatedcytosinesareconvertedtouracilswhile5-methylcytosinesremainunaltered.Thus,thesequenceofthetreatedDNAwilldifferiftheDNAisoriginallymethylatedvs.unmethylated.PrimerscontainedintheCpGWIZ®RB1AmplificationKitaredesignedtospecificallyamplifyeachofthesequencesbaseduponthesechemically-induceddifferences.IfthesampleDNAwasoriginallyunmethylated,aproductwillbegeneratedafterPCRusingtheUprimerset.Conversely,aproductwillbegeneratedusingtheMprimersetifthesamplewasoriginallymethylated.
BackgroundInformation	Methylationofcytosineslocated5"toguanosineisknowntohaveaprofoundeffectontheexpressionofseveraleukaryoticgenes(1).Innormalcells,methylationoccurspredominantlyinCG-poorregions,whileCG-richareas,calledCpGislands,remainunmethylated.AberrantmethylationofnormallyunmethylatedCpGislandshasbeendocumentedasarelativelyfrequenteventinimmortalizedandtransformedcells(2)andhasbeenassociatedwithtranscriptionalinactivationofdefinedtumorsuppressorgenesinhumancancers(3,4).Theretinoblastoma1(RB1)geneexhibitscharacteristichypermethylationimmanymaligninanttumors. PreviouslydevelopedmethodstodeterminethemethylationstatusofcytosineincludedigestionwithmethylationsensitiverestrictionenzymesandgenomicDNAsequencing.Bothtechniqueshavelimitations:restrictionenzymescanonlydetectmethylationsiteswithintheirrecognitionsequenceandsequencingistimeconsuming.IncreasingthedetectionsensitivityofCpGislandmethylationhasthepotentialtodefinetumorsuppressorgenefunctionandprovidesanewstrategyforearlytumordetection. Methylation-specificPCR(MSP)isanewtechnologyforsensitivedetectionofabnormalgenemethylationutilizingsmallamountsofDNA(5).ThisprocessemploysaninitialbisulfitereactiontomodifytheDNA,followedbyPCRamplificationwithspecificprimersdesignedtodistinguishmethylatedfromunmethylatedDNA.TheCpGenome™DNAModificationKit(S7820)containsthereagentsnecessarytoperformtheinitialbisulfitereactions,whiletheCpGWIZ®RB1AmplificationKitcontainsthereagentsrequiredforthePCRamplificationreactions.
MaterialsRequiredbutNotDelivered	EquipmentandSupplies a.MicrocentrifugetubesforPCRamplification b.Aerosol-resistantPipettetips c.Thermocycler d.Gelelectrophoresisapparatus(verticalorhorizontal) e.PowerSupply f.302nmUVtransIlluminator,cameraandfilm Reagents a2.5mMdNTPmix(2.5mMofeachnucleotide) b."Hotstart"Taqpolymerase c."Hotstart"PCRreagents(seeSec.II.Protocols). d.Reagentsforgelelectrophoresis(1XTBEand2%agarose,10%acrylamide,orsuitablehighresolutionagarose) e.DNAMarkers(sizerange100-300bp) f.Ethidiumbromide(10mg/mL) g.Gel-loADIngsolution/LoadingDye h.BisulfiteModifiedDNA(CpGenome™DNAModificationKit,S7820)

ProductInformation

Components

ThecomponentsoftheCpGWIZ®RB1AmplificationKitincludethoserequiredforPCRamplificationafterbisulfitemodificationofDNAsamples.Sufficientreagentsareprovidedtoanalyze25sampleswithappropriatecontrols. UPrimerSet7.5μMeachprimer(25X)35μL(neutralcap)90552-15°Cto-25°C MPrimerSet7.5μMeachprimer(25X)35μL(redcap)90553-15°Cto-25°C WPrimerSet7.5μMeachprimer(25X)35μL(greencap)90554-15°Cto-25°C UcontrolDNA0.1μg/μL50μL(whitecap)90393-15°Cto-25°C McontrolDNA0.1μg/μL50μL(redcap)90394-15°Cto-25°C WcontrolDNA0.05μg/μL50μL(greencap)90395-15°Cto-25°C Universal10XPCRBuffer265μL(bluecap)90396-15°Cto-25°C

StorageandShippingInformation

Applications
Application	MSP,performedusingtheCpGenomeDNAModificationKit&theCpGWIZRB1AmplificationKit,permitssensitivedetectionofalteredDNA.
KeyApplications	PCR

BIOLOGicalInformation
SpeciesReactivity	Human
EntrezGeneNumber	NM_000321.2
EntrezGeneSummary	Retinoblastoma(RB)isanembryonicmalignantneoplasmofretinalorigin.Italmostalwayspresentsinearlychildhoodandisoftenbilateral.Spontaneousregression("cure")occursinsomecases.TheretinoblastomageneRBwasthefirsttumorsuppressorgenecloned,andisanegativeregulatorofthecellcyclethroughitsABIlitytobindthetranscriptionfactorE2F(MIM189971)andrepresstranscriptionofgenesrequiredforSphase(HanahanandWeinberg,2000[PubMed10647931]).[suppliedbyOMIM]
GeneSymbol	RB1 P105-RB retinoblastoma-1 RB PP110 OSRC
UniProtNumber	P06400
UniProtSummary	FUNCTION:SwissProt:P06400#Keyregulatorofentryintocelldivisionthatactsasatumorsuppressor.Directlyinvolvedinheterochromatinformationbymaintainingoverallchromatinstructureand,inparticular,thatofconstitutiveheterochromatinbystabilizinghistonemethylation.RecruitsandtargetshistonemethyltransferasesSUV39H1,SUV420H1andSUV420H2,leadingtoepigenetictranscriptionalrepression.ControlshistoneH4"Lys-20"trimethylation.AlsoactsasatranscriptionrepressorofE2Ftargetgenesbyrecruitingchromatin-modifyingenzymestopromoters.InhibitstheintrinsickinaseactivityofTAF1.FormsacomplexwithadenovirusE1AandwithSV40largeTantigen.MaybindandmodulatefunctionallycertaincellularproteinswithwhichTandE1Acompeteforpocketbinding. SIZE:928aminoacids;106159Da SUBUNIT:InteractspreferentiallywithtranscriptionfactorE2F1.TheunphosphorylatedforminteractswithARID3B,JARID1A,SUV39H1,MJD2A/JHDM3AandTHOC1.InteractswiththeN-terminaldomainofTAF1.InteractswithAATF,DNMT1,LIN9,LMNA,SUV420H1,SUV420H2,PELP1andTMPO-alpha.MayinteractwithKNTC2.InteractswithEID1andZUBR1.InteractswithARID4AandJARID1B. SUBCELLULARLOCATION:Nucleus. TISSUESPECIFICITY:Expressedintheretina. PTM:PhosphorylatedfromStoMphaseofthecellcycleandisdephosphorylatedinG1.T,butnotE1A,bindsonlytotheunphosphorylatedform. DISEASE:SwissProt:P06400#DefectsinRB1arethecauseofchildhoodcancerretinoblastoma(RB)[MIM:180200].RBisacongenitalmalignanttumorthatarisesfromthenuclearlayersoftheretina.Itoccursinabout1:20"000livebirthsandrepresentsabout2%ofchildhoodmalignancies.Itisbilateralinabout30%ofcases.AlthoughmostRBappearsporadically,about20%aretransmittedasanautosomaldominanttraitwithincompletepenetrance.Thediagnosisisusuallymadebeforetheageof2yearswhenstrabismusoragraytoyellowreflexfrompupil(cateye)isinvestigated.&DefectsinRB1areacauseofbladdercancer[MIM:109800].&DefectsinRB1areacauseofosteogenicsarcoma[MIM:259500]. SIMILARITY:SwissProt:P06400##Belongstotheretinoblastomaprotein(RB)family.

PhysicochemicalInformation

Dimensions

MaterialsInformation

MaterialsInformation

Millipore 提供三种不同的 Immobilon PVDF 膜，是针对不同的蛋白质印迹应用的理想选择。 现在我们还 提供采用按规格裁切的印迹方片膜和两层过滤纸组成的印迹三明治。   与硝酸纤维素膜相比，Immobilon 膜具有多种优点。 它们不易破裂或卷 曲，便于裁剪且在切割过程中不会碎裂。 它们还有低背景、广泛溶剂相容性以及优异的着色能力。 此外，它们可以重复检测。 Immobilon-P 膜（沃比森-供应） ·         0.45μm 孔径 ·         建议用于大多数免疫印迹，特别在蛋 白质大于 20kDa 时 ·         Millipore 的快速免疫检测实验方法不需要膜封闭并缩短洗涤时间，将检测时间至少减少了两个小时，而不致降低灵敏 度或特异性 用于高通量实验室的印迹三明治 ·         当您需要处理多个印迹时，印迹三明治是一种便利省时的选择。 我们的印迹三明 治包含多片 Immobilon-P 膜，其中插入了按规格裁切的多张层析级印迹纸。 转印滤纸 ·         按照大多数免疫印迹尺寸进行裁切的层析级转印滤纸。 Immobilon-PSQ 膜（沃比森-供应） ·         0.2μm 孔径和大的内表面面积 ·         较其它膜具有更高的 蛋白质吸附率和测序得率 ·         建议用于小于 20kDa 的印迹蛋白质 ·         防止小分子量的蛋白质的渗漏 Immobilon-FL 膜（沃比森-供应） ·        针对荧光应用进行优化的转印膜 ·         极低的背景提高了所 有荧光检测实验的灵敏度 ·         与所有常用的荧光探针（所有激发和发射波长）兼容 ·         是多态性检测和化学荧光应用的理想选择