![StressMarq/StressXpress® Cyclic GMP CLIA Kit (High-Sensitivity)/SKT-210-96/96-well](images/StressMarq/SKT-210_Cyclic_GMP_CLIA_Kit_High-Sensitivity_Regular_Format_Standard_Curve_Fig1.png)
Overview:
ProductName | CyclicGMPCLIAKit(High-Sensitivity) |
Description | ChemiluminescentdetectionofcGMP |
SpeciesReactivity | SpeciesIndependent |
Platform | Microplate |
SampleTypes | Celllysates,EDTAPlasma,Saliva,Tissue,TissueCultureMedia,Urine |
DetectionMethod | ChemiluminescentAssay |
AssayType | DirectCLIA(ChemiluminescentImmunoassay) |
Utility | CLIAkitusedtomeasurecGMPpresentinsamples. |
Sensitivity | 0.034pmol/ml |
AssayRange | Regular:0.0469-3pmol/ml.Acetylated:0.03125-1pmol/ml |
Precision | IntraAssayPrecision(Regular):TwohumanurinesamplesweredilutedwithSampleDiluentandruninreplicatesof22inanassay.ThemeanandprecisionofthecalculatedcGMPconcentrationswere:Sample1-0.053pmol/mL,10.6%CVSample2-0.225pmol/mL,11.8%CVIntraAssayPrecision(Acetylated):TwohumanurinesamplesweredilutedwithSampleDiluent,acetylatedandruninreplicatesoftwenty-twoinanassay.ThemeanandprecisionofthecalculatedcGMPconcentrationswere:Sample1-0.032pmol/mL,8.9%CVSample2-0.018pmol/mL,8.3%CVInterAssayPrecision(Regular):TwohumanurinesamplesweredilutedwithSampleDiluentandruninduplicatesinfourteenassaysrunovermultipledaysbyfouroperators.ThemeanandprecisionofthecalculatedcGMPconcentrationswere:Sample1-0.213pmol/mL,14.9%CVSample2-0.083pmol/mL,18.0%CVInterAssayPrecision(Acetylated):TwohumanurinesampleweresdilutedwithSampleDiluent,acetylatedandruninduplicatesineightassaysrunovermultipledaysbyfouroperators.ThemeanandprecisionofthecalculatedcGMPconcentrationswere:Sample1-0.07pmol/mL,15.1%CVSample2-0.04pmol/mL,15.1%CV |
NumberofSamples | 39samplesinduplicate |
OtherResources | KitBooklet,MSDS,SalivaSampleHandlingInstructions |
Properties
StorageTemperature | 4ºC | ||||||||||||||||||||||||||||||||||||||||||
ShippingTemperature | BlueIce | ||||||||||||||||||||||||||||||||||||||||||
ProductType | CLIAKits | ||||||||||||||||||||||||||||||||||||||||||
AssayOverview | TheCyclicGMP(cGMP)CLIA(High-Sensitivity)kitisdesignedtoquantitativelymeasurecGMPpresentinlysedcells,EDTAplasma,urine,salivaandtissueculturemediasamples.ForsampleswherethelevelsofcGMPareexpectedtoberelativelyhigh,theregularformatfortheassaycanbeused.ForsampleswithexpectedlowlevelsofcGMP,anoptionalacetylationprotocolcanbeused.ThekitisuniqueinthatallsamplesandstandardsaredilutedintoanacidicSampleDiluent,whichcontainsspecialadditivesandstABIlizers,forcGMPmeasurement.Thisallowsplasma,urineandsalivasamplestobereadinanidenticalmannertolysedcells.AcidifiedsamplesofcGMParestableandendogenousphosphodiesterasesareinactivatedintheSampleDiluent.AcGMPstandardisprovidedtogenerateastandardcurvefortheassayandallsamplesshouldbereadoffthestandardcurve.AwhitemicrotiterplatecoatedwithanantibodytocapturemouseIgGisprovidedandaneutralizingPlatePrimersolutionisaddedtoalltheusedwells.Standardsordilutedsamples,eitherwithorwithoutacetylation,arePipettedintotheprimedwells.AcGMP-peroxidaseconjugateisaddedtothestandardsandsamplesinthewells.ThebindingreactionisinitiatedbytheadditionofamousemonoclonalantibodytocGMPtoeachwell.Afteranovernightincubationat4°C,theplateiswashedandthechemiluminescentsubstrateisadded.ThesubstratereactswiththeboundcGMP-peroxidaseconjugatetoproducelightThegeneratedlightisdetectedinamicrotiterplatereadercapableofreADIngluminescence.TheconcentrationofthecGMPinthesampleiscalculated,aftermakingsuitablecorrectionforthedilutionofthesample,usingsoftwareavailablewithmostplatereaders. | ||||||||||||||||||||||||||||||||||||||||||
KitOverview |
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CiteThisProduct | CyclicGMPCLIAKit(High-Sensitivity)(StressMarqBiosciencesInc.,VictoriaBCCANADA,Catalog#SKT-210) |
BIOLOGicalDescription
AlternativeNames | cGMP;3",5"-cyclicGMPCLIAKit,GuanosinecyclicmonophosphateCLIAKit,Cyclic3",5"-GMP;Guanosine3",5"-cyclicphosphateCLIAKit |
ResearchAreas | CellSignaling,Neuroscience |
ScientificBackground | Guanosine3’,5’-cyclicmonophosphate(cyclicGMP;cGMP)isacriticalandmultifunctionalsecondmessengerpresentatlevelstypically10-100foldlowerthancAMPinmosttissues.IntracellularcGMPisformedbytheactionoftheenzymeguanylatecyclaseonGTPanddegradedthroughphosphodiesterasehydrolysis(1-3).Guanylatecyclases(GC)areeithersolubleormembranebound(3,4).SolubleGCsarenitricoxideresponsive,whereasmembraneboundGCsrespondtohormonessuchasacetylcholine,insulinandoxytocin.OtherchemicalslikeSEROtoninandhistaminealsocauseanincreaseincGMPlevels(5,6).CyclicGMPregulatescellularcompositionthroughcGMP-dependentkinase,cGMP-dependentionchannelsortransporters,andthroughitshydrolyticdegradationbyphosphodiesterase(1,7) |
References | 1.Domek-Lopacinska,K.andStrosznajder,JB.(2005)JPhysiolPharmacol56Suppl2,15-34. 2.Lucas,K.A.etal.(2000)PharmacolRev52:375-414. 3.Ashman,DF.,etal.(1963),BiochemBiophysResComm,11:330-4. 4.PotterLR,Abbey-HoschS,andDickeyDM.(2006)EndocrRev27:47-72. 5.Waldman,SAandMurad,F.(1987)PharmacolRevs,39:163-197. 6.TremblayJ,GerzerR,andHametP.(1988),Adv.2ndMessanger&PhosphoproteinRes.,22:319-383. 7.MatsumotoT,KobayashiT,andKamataK.(2003)JSmoothMuscleRes39:67-86. |
ProductImages
![<p>Regular Format Typical Standard Curve for the Cyclic GMP CLIA Kit (High-Sensitivity) (Chemiluminescent Immunoassay) StressXpress® – SKT-210. Assay Type: Direct CLIA. Detection Method: Chemiluminescent Assay. Assay Range: Regular: 0.0469 – 3 pmol/ml. Acetylated: 0.03125 – 1 pmol/ml.</p>](images/StressMarq/201710/SKT-210_Cyclic_GMP_CLIA_Kit_High-Sensitivity_Regular_Format_Standard_Curve_Fig1.png)
RegularFormatTypicalStandardCurvefortheCyclicGMPCLIAKit(High-Sensitivity)(ChemiluminescentImmunoassay)StressXpress®–SKT-210.AssayType:DirectCLIA.DetectionMethod:ChemiluminescentAssay.AssayRange:Regular:0.0469–3pmol/ml.Acetylated:0.03125–1pmol/ml.
![<p>Acetylated Format Typical Standard Curve for the Cyclic GMP CLIA Kit (High-Sensitivity) (Chemiluminescent Immunoassay) StressXpress® – SKT-210. Assay Type: Direct CLIA. Detection Method: Chemiluminescent Assay. Assay Range: Regular: 0.0469 – 3 pmol/ml. Acetylated: 0.03125 – 1 pmol/ml.</p>](images/StressMarq/201710/SKT-210_Cyclic_GMP_CLIA_Kit_High-Sensitivity_Acetylated_Format_Standard_Curve_Fig3.png)
AcetylatedFormatTypicalStandardCurvefortheCyclicGMPCLIAKit(High-Sensitivity)(ChemiluminescentImmunoassay)StressXpress®–SKT-210.AssayType:DirectCLIA.DetectionMethod:ChemiluminescentAssay.AssayRange:Regular:0.0469–3pmol/ml.Acetylated:0.03125–1pmol/ml.
![<p>Linearity was determined by taking two human urine samples, one with a low cGMP level of 0.3 pmol/mL and one with a higher level of 9.7 pmol/mL, and mixing them in ratios. The measured concentrations were compared to the expected values based on the ratios used.</p>](images/StressMarq/201710/SKT-210_Cyclic_GMP_CLIA_Kit_High-Sensitivity_Linearity_Recovery_Graph_Fig4.png)
Linearitywasdeterminedbytakingtwohumanurinesamples,onewithalowcGMPlevelof0.3pmol/mLandonewithahigherlevelof9.7pmol/mL,andmixingtheminratios.Themeasuredconcentrationswerecomparedtotheexpectedvaluesbasedontheratiosused.
![<p>Chemical structure of Cyclic GMP (Cyclic guanosine monophosphate)</p>](images/StressMarq/201710/SKT-210_Cyclic_GMP_CLIA_Kit_High-Sensitivity_Chemical_Structure_of_cGMP_Fig1.png)
ChemicalstructureofCyclicGMP(Cyclicguanosinemonophosphate)
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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!
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