Overview:
ProductName | CyclicAMPCLIAKit(High-Sensitivity) |
Description | ChemiluminescentdetectionofcAMP |
SpeciesReactivity | SpeciesIndependent |
Platform | Microplate |
SampleTypes | Celllysates,EDTAPlasma,HeparinPlasma,Saliva,Tissue,TissueCultureMedia,Urine |
DetectionMethod | ChemiluminescentAssay |
AssayType | DirectCLIA(ChemiluminescentImmunoassay) |
Utility | CLIAkitusedtoquantitativelymeasurecAMPpresentinsamples. |
Sensitivity | 0.119pmol/ml |
AssayRange | Regular:0.185-15pmol/ml.Acetylated:0.039-5pmol/ml |
Precision | IntraAssayPrecision(Regular):TwohumanurinesamplesweredilutedwithSampleDiluentandruninreplicatesof20inanassay.ThemeanandprecisionofthecalculatedcAMPconcentrationswere:Sample1-11.2pmol/mL,3.9%CVSample2-3.6pmol/mL,8.3%CVIntraAssayPrecision(Acetylated):TwohumanplasmasamplesweredilutedwithSampleDiluent,acetylatedandruninreplicatesof20inanassay.ThemeanandprecisionofthecalculatedcAMPconcentrationswere:Sample1-1.32pmol/mL,8.4%CVSample2-0.39[pmol10.9%CVInterAssayPrecision(Regular):TwohumanurinesamplesweredilutedwithSampleDiluentandruninduplicatesinfourteenassaysrunovermultipledaysbyfouroperators.ThemeanandprecisionofthecalculatedcAMPconcentrationswere:Sample1-6.5pmol/mL,8.1%CVSample2-2.1pmol/mL,13.0%CVInterAssayPrecision(Acetylated):TwohumanplasmasampleweredilutedwithSampleDiluent,acetylatedandruninduplicatesinfourteenassaysrunovermultipledaysbyfouroperators.ThemeanandprecisionofthecalculatedcAMPconcentrationswere:Sample1-1.02pmol/mL,10.1%CVSample2-0.32pmol/mL,16.5%CV |
IncubationTime | 2hours |
NumberofSamples | 38samplesinduplicate |
OtherResources | KitBooklet,MSDS,SalivaSampleHandlingInstructions |
Properties
StorageTemperature | 4ºC | ||||||||||||||||||||||||||||||||||||||||||
ShippingTemperature | BlueIce | ||||||||||||||||||||||||||||||||||||||||||
ProductType | CLIAKits | ||||||||||||||||||||||||||||||||||||||||||
AssayOverview | TheCyclicAMP(cAMP)CLIA(High-Sensitivity)kitisdesignedtoquantitativelymeasurecAMPpresentinlysedcells,EDTAandheparinplasma,urine,salivaandtissueculturemediasamples.Fortissuesamples,salivaandurine,wherethelevelsofcAMPareexpectedtoberelativelyhigh,theregularformatfortheassaycanbeused.Forplasmasamplesandsomedilutecelllysatesanoptionalacetylationprotocolcanbeused.Thiskitcanmeasureaslittleas1femtomolcAMPpersample.ThekitisuniqueinthatallsamplesandstandardsaredilutedintoanacidicSampleDiluent,whichcontainsspecialadditivesandstABIlizers,forcAMPmeasurement.Thisallowsplasma,urineandsalivasamplestobereadinanidenticalmannertolysedcells.AcidifiedsamplesofcAMParestableandendogenousphosphodiesterasesareinactivatedintheSampleDiluent.AcAMPstandardisprovidedtogenerateastandardcurvefortheassayandallsamplesshouldbereadoffthestandardcurve.AwhitemicrotiterplatecoatedwithanantibodytocapturesheepIgGisprovided.PriortotheadditionofanysamplesorstandardsaneutralizingPlatePrimersolutionisaddedtoalltheusedwells.Standardsordilutedsamples,eitherwithorwithoutacetylation,arePipettedintotheprimedwells.AcAMP-peroxidaseconjugateisaddedtothestandardsandsamplesinthewells.ThebindingreactionisinitiatedbytheadditionofasheepantibodytocAMPtoeachwell.Aftera2hourincubation,theplateiswashedandthechemiluminescentsubstrateisadded.ThesubstratereactswiththeboundcAMP-peroxidaseconjugatetoproducelightThegeneratedlightisdetectedinamicrotiterplatereadercapableofreADIngluminescence.TheconcentrationofthecAMPinthesampleiscalculated,aftermakingsuitablecorrectionforthedilutionofthesample,usingsoftwareavailablewithmostplatereaders. | ||||||||||||||||||||||||||||||||||||||||||
KitOverview |
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CiteThisProduct | CyclicAMPCLIAKit(High-Sensitivity)(StressMarqBiosciencesInc.,VictoriaBCCANADA,Catalog#SKT-208) |
BIOLOGicalDescription
AlternativeNames | 3"-5"-cyclicadenosinemonophosphateCLIAKit |
ResearchAreas | CellSignaling,Neuroscience |
ScientificBackground | Adenosine-3’,5’-cyclicmonophosphate(cyclicAMP)isasecondmessengerthatplaysanimportantroleinintracellularregulation.Specifically,itfunctionsasamediatorofactivityofseveralkeyhormonesincludingepinephrine,glucagon,andACTH(1-4).AdenylatecyclaseisactivatedbythehormonesglucagonandadrenalineandbyGprotein.Liveradenylatecyclaserespondsmorestronglytoglucagon,andmuscleadenylatecyclaserespondsmorestronglytoadrenaline.cAMPdecompositionintoAMPiscatalyzedbytheenzymephosphodiesterase.IntheHumanMetabolomeDatabasethereare166metabolicenzymeslistedthatconvertcAMP(5).OtherbiologicalactionsofcAMPincluderegulationofinnateimmunefunctioning(6),axonregeneration(7),cancer(8),andinflammation(9). |
References | 1.Sutherland,E.W.andRall,T.W.FractionationandCharacterizationofaCyclicAdenineRibonucleotideFormedbyTissueParticles.J.Biol.Chem.,232:1077,1958. 2.Marsh,J.M.Biol.Reprod.,14:30-53,1976. 3.Korenman,S.G.andKrall,J.F.Biol.Reprod.,16:1-17,1977. 4.Kelley,D.J.,Bhattacharyya,A.,Lahvis,G.P.,Yin,J.C.P.,Malter,J.,andDavidson,R.J.Neurosci.Biobehav.Rev.,32(8):1533-1543,2008. 5.http://www.hmdb.ca/metabolites/HMDB00058 6.Serezani,C.H.,Ballinger,M.N.,Aronoff,D.M.,andPeters-Golden,M.Am.J.Resp.CellandMol.Biol.,39(2):127,2008. 7.Hannila,S.S.,andFilbin,M.T.Exp.Neurol.,209(2):321–332,2008. 8.Shankar,D.B,Cheng,J.C.,andSakamoto,K.M.Cancer,104(9):1819-24,2005. 9.GaleaE.andFeinstein,D.L.FASEBJ.,13:2125-2137,1999. |
ProductImages
RegularFormatTypicalStandardCurvefortheCyclicAMPCLIAKit(High-Sensitivity)(ChemiluminescentImmunoassay)StressXpress®–SKT-208.AssayType:DirectCLIA.DetectionMethod:ChemiluminescentAssay.AssayRange:Regular:0.185–15pmol/ml.Acetylated:0.039–5pmol/ml.
AcetylatedFormatTypicalStandardCurvefortheCyclicAMPCLIAKit(High-Sensitivity)(ChemiluminescentImmunoassay)StressXpress®–SKT-208.AssayType:DirectCLIA.DetectionMethod:ChemiluminescentAssay.AssayRange:Regular:0.185–15pmol/ml.Acetylated:0.039–5pmol/ml.
LinearitywasdeterminedbytakingtwoJurkatcelllysatesamples,onewithalowcAMPlevelof0.2pmol/mLandonewithahigherlevelof2.5pmol/mL,andmixingthemintheratios.Themeasuredconcentrationswerecomparedtotheexpectedvaluesbasedontheratiosused.
ChemicalstructureofCyclicAMP(cAMP)
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TAE是使用最广泛的缓冲系统。其特点是超螺旋在其中电泳时更符合实际相对分子质量(TBE中电泳时测出的相对分子质量会大于实际分子质量),且双链线状DNA在其中的迁移率较其他两种缓冲液快约10%,电泳大于13kb的片段时用TAE缓冲液将取得更好的分离效果,此外,回收DNA片段时也易用TAE缓冲系统进行电泳。TAE的缺点是缓冲容量小,长时间电泳(如过夜)不可选用,除非有循环装置使两极的缓冲液得到交换。 50×TAE Buffer 配制方法: 1. 称量Tris 242g,Na2EDTA.2H2O 37.2g 于1L烧杯中; 2.向烧杯中加入约800ml去离子水,充分搅拌均匀; 3加入57.1ml的冰乙酸,充分溶解; 4.加去离子水定容至1L后,室温保存。向左转|向右转
低离子强度时,迁移率快。但离子强度过低,缓冲液的缓冲容量小,不易维持pH恒定。高离子强度时迁移率慢,因此为了获得更快的反应速度,在缓冲容量允许的范围内,离子强度应该尽可能小。
Tris-Glycine/SDS;
MOPS/SDS;
Bis-Glycine/SDS等不同的缓冲液、预混液等。
不知道这几种缓冲液有什么异同呢?
比如如果是Invitrogen的预制胶-预混液电泳系统,每种预制胶有适配缓冲液,但是如果用其他的缓冲液似乎也可以得到不错的结果,那么是否说明电泳缓冲液只要满足了缓冲ph范围,其他的不是特别重要呢?
此外,附加提问是,PVDF膜在转膜的时候,转膜缓冲液中不需要加入甲醇,那么是加入甲醇转膜效果好还是不加好呢?
谢谢!