
Overview:
ProductName | CyclicAMPEIAKit |
Description | QuantitativecolorimetricdetectionofcAMP |
SpeciesReactivity | SpeciesIndependent |
Platform | Microplate |
SampleTypes | Celllysates,EDTAPlasma,HeparinPlasma,Saliva,Tissue,TissueCultureMedia,Urine |
DetectionMethod | ColorimetricAssay |
AssayType | DirectEIA(EnzymeImmunoassay) |
Utility | EIAkitusedtoquantitativelymeasurecAMPpresentinsamples. |
Sensitivity | 0.64pmol/ml |
AssayRange | Regular:0.617-150pmol/ml.Acetylated:0.156-10pmol/ml |
Precision | IntraAssayPrecision(Regular):ThreehumanurinesamplesweredilutedwithSampleDiluentandruninreplicatesof20inanassay.ThemeanandprecisionofthecalculatedcAMPconcentrationswere:Sample1-56.9pmol/mL,8.6%CVSample2-11.9pmol/mL,11.3%CVSample3-6.7pmol/mL,12.3%CVIntraAssayPrecision(Acetylated):TwohumanplasmasamplesweredilutedwithSampleDiluent,acetylatedandruninreplicatesof20inanassay.ThemeanandprecisionofthecalculatedcAMPconcentrationswere:Sample1-1.74pmol/mL,10.4%CVSample2-0.60pmol/mL,11.8%CVInterAssayPrecision(Regular):ThreehumanurinesamplesweredilutedwithSampleDiluentandruninduplicatesintwelveassaysrunovermultipledaysbyfouroperators.ThemeanandprecisionofthecalculatedcAMPconcentrationswere:Sample1-57.1pmol/mL,10.0%CVSample2-10.9pmol/mL,11.5%CVSample3-6.3pmol/mL,11.3%CVInterAssayPrecision(Acetylated):OnehumanurineandtwohumanplasmasampleweredilutedwithSampleDiluent,acetylatedandruninduplicatesintwelveassaysrunovermultipledaysbyfouroperators.ThemeanandprecisionofthecalculatedcAMPconcentrationswere:Sample1-5.22pmol/mL,9.8%CVSample2-0.99pmol/mL,8.1%CVSample3-0.73pmol/mL,15.4%CV |
IncubationTime | 2.5Hours |
NumberofSamples | 39samplesinduplicate |
OtherResources | KitBooklet,MSDS,SalivaSampleHandlingInstructions |
Properties
StorageTemperature | 4ºC | |||||||||||||||||||||||||||||||||||||||
ShippingTemperature | BlueIce | |||||||||||||||||||||||||||||||||||||||
ProductType | EIAKits | |||||||||||||||||||||||||||||||||||||||
AssayOverview | TheCyclicAMPEIAkitisdesignedtoquantitativelymeasurecAMPpresentinlysedcells,EDTAandheparinplasma,urine,salivaandtissueculturemediasamples.Fortissuesamples,salivaandurine,wherethelevelsofcAMPareexpectedtoberelativelyhigh,theregularformatfortheassaycanbeused.Forplasmasamplesandsomedilutecelllysatesanoptionalacetylationprotocolcanbeused.ThekitisuniqueinthatallsamplesandstandardsaredilutedintoanacidicSampleDiluent,whichcontainsspecialadditivesandstABIlizers,forcAMPmeasurement.Thisallowsplasma,urineandsalivasamplestobereadinanidenticalmannertolysedcells.AcidifiedsamplesofcAMParestableandendogenousphosphodiesterasesareinactivatedintheSampleDiluent.AcAMPstandardisprovidedtogenerateastandardcurvefortheassayandallsamplesshouldbereadoffthestandardcurve.AclearmicrotiterplatecoatedwithanantibodytocapturesheepIgGisprovidedandaneutralizingPlatePrimersolutionisaddedtoalltheusedwells.Standardsordilutedsamples,eitherwithorwithoutacetylation,arePipettedintotheprimedwells.AcAMP-peroxidaseconjugateisaddedtothestandardsandsamplesinthewells.ThebindingreactionisinitiatedbytheadditionofasheepantibodytocAMPtoeachwell.Aftera2hourincubation,theplateiswashedandsubstrateisadded.ThesubstratereactswiththeboundcAMP-peroxidaseconjugate.Afterashortincubation,thereactionisstoppedandtheintensityofthegeneratedcolorisdetectedinamicrotiterplatereadercapableofmeasuring450nmwavelength.TheconcentrationofthecAMPinthesampleiscalculated,aftermakingsuitablecorrectionforthedilutionofthesample,usingsoftwareavailablewithmostplatereaders. | |||||||||||||||||||||||||||||||||||||||
KitOverview |
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CiteThisProduct | CyclicAMPEIAKit(StressMarqBiosciencesInc.,VictoriaBCCANADA,Catalog#SKT-209) |
BIOLOGicalDescription
AlternativeNames | 3"-5"-cyclicadenosinemonophosphateEIAKit |
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

RegularFormatTypicalStandardCurvefortheCyclicAMPEIAKit(EnzymeImmunoassay)StressXpress®–SKT-209.AssayType:DirectEIA.DetectionMethod:ColorimetricAssay.AssayRange:Regular:0.617–150pmol/ml.Acetylated:0.156–10pmol/ml.

AcetylatedFormatTypicalStandardCurvefortheCyclicAMPEIAKit(EnzymeImmunoassay)StressXpress®–SKT-209.AssayType:DirectEIA.DetectionMethod:ColorimetricAssay.AssayRange:Regular:0.617–150pmol/ml.Acetylated:0.156–10pmol/ml.

ChemicalstructureofCyclicAMP(Adenosine-3,5-cyclicmonophosphate)
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② 应取pH8.0,这样可使核苷酸带较多负电荷,利于吸附于阴离子交换树脂柱。虽然pH 11.4时核苷酸带有更多的负电荷,但pH过高对分离不利。
③ 当不考虑树脂的非极性吸附时,根据核苷酸负电荷的多少来决定洗脱速度,则洗脱顺序为CMP>AMP> GMP > UMP,但实际上核苷酸和聚苯乙烯阴离子交换树脂之间存在着非极性吸附,嘌呤碱基的非极性吸附是嘧啶碱基的3倍。静电吸附与非极性吸附共同作用的结果使洗脱顺序为:CMP> AMP > UMP >GMP。
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。
2.配好的EDTA不灭菌行不?放4度冰箱等过完年回来直接拿去配TAE
3.TAE配好后不用灭菌吧
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
但是最好还是不要用,影响实验就不好了。

