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主营:研究并生产荧光和发光探针,信号转导研究的试剂
℡ 4000-520-616
℡ 4000-520-616
AAT Bioquest/Amplite™ Fluorimetric Neuraminidase Assay Kit *Blue Fluorescence*/12602/200 Tests
产品编号:12602
市  场 价:¥85560.00
场      地:美国(厂家直采)
联系QQ:1570468124
电话号码:4000-520-616
邮      箱: info@ebiomall.com
美  元  价:$4278.00
品      牌: AAT Bioquest
公      司:AAT Bioquest
公司分类:
AAT Bioquest/Amplite™ Fluorimetric Neuraminidase Assay Kit *Blue Fluorescence*/12602/200 Tests
商品介绍
Overview
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Ex/Em(nm)360/449
MWN/A
CAS#N/A
SolventN/A
StorageF/D/L
CategoryEnzymeDetection
HydrolyticEnzymes
RelatedDiagnosticMolecules
BiochemicalAssays
Neuraminidases,alsocalledsialidases,areglycosidehydrolaseenzymesthatcatalyzethehydrolysisofterminalsialicacidresiduesandneuraminicacid.Themostcommonlyknownneuraminidaseistheviralneuraminidase.Thecleavageoflinkagebetweensialicacidandadjacentsugarresiduepermitsthetransportofthevirusthroughmucinanddestroysthehaemagglutininreceptoronthehostcell,thusallowingelutionofProgenyvirusparticlesfrominfectedcells.Neuraminidasepromotesinfluenzavirusreleasefrominfectedcellsandfacilitatesvirusspreadwithintherespiratorytract.Thus,itisanimportanttargetforinfluenzadrugdevelopment.ThedetectionofneuraminidaseandscreeningitsinhibitorsisoneoftheessentialtasksforinvestigatingBIOLOGicalprocessesandpreventionofinfluenzainfection.Thereareafewassaykitsavailablefordetectingneuraminidase,butallthecommercialavailablekitsaretedioustouse.OurAmplite™FluorimetricNeuraminidaseAssayKitprovidesasensitiveandrobustfluorimetricassaytodetectneuraminidasethatexistseitherincellsorbiologicalsamples.Thenon-fluorescentneuraminidasesubstratebecomesstronglyfluorescentuponneuraminidasecleavage.Thekitcandetectaslittleas0.3mU/mLneuraminidaseina100µLassayvolume.Theassaycanbeperformedinaconvenient96-wellor384-wellmicrotiter-plateformatandeasilyadaptedtoautomationwithoutaseparationstep.ThesignalcanbeeasilyreadbyafluorescencemicroplatereaderatEx/Em=~320/~450nm.

Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.Prepareneuraminidasestandardstocksolution:

Add50µLofddH2OintothevialofNeuraminidaseStandard(ComponentC)tomake2U/mLneuraminidasestandardstocksolution.

Note:Theconcentrationofthisstocksolutionisapproximately2U/mL.TheunusedNeuraminidaseStandardsolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.

 

2.Prepare200XFluLiteTMBluestocksolution:

Add50µLofddH2OintothevialofFluLiteTMBlue(ComponentA)tomake200Xstocksolution.

Note:TheunusedFluLiteTMBluesolutionshouldbedividedintosingleusealiquotsandstoredat-20oCandkeptfromlight.

 

3.Prepareneuraminidaseassaymixture:

Add25µLof200XFluLiteTMBluestocksolution(fromStep2)into5mLofAssayBuffer(ComponentB),andmixwell.

 

4.Prepareserialdilutionsofneuraminidasestandard(0to20mU/mL):

4.1   Add10μLof2U/mLneuraminidasestandardstocksolution(fromStep1)to990µLofassaybuffer(ComponentB)togenerate20mU/mLneuraminidasestandard.

Note:Dilutedneuraminidasestandardsolutionisunstable.Usewithin4hours.

 

4.2   Take500μLof20mU/mLneuraminidasestandardsolution(fromStep4.1)toperform1:2serialdilutionstoget10,5,2.5,1.25,0.625,0.312and0mU/mLserialdilutionsofneuraminidasestandard.

 

4.3   Addneuraminidasestandardsandneuraminidase-containingtestsamplesintoasolidblack96-wellmicroplateasshowninTables1and2.

 

Table1Layoutofneuraminidasestandardsandtestsamplesinasolidblack96-wellmicroplate

BL

BL

TS

TS

….

….

 

 

 

 

 

 

NA1

NA1

….

….

….

….

 

 

 

 

 

 

NA2

NA2

 

 

 

 

 

 

 

 

 

 

NA3

NA3

 

 

 

 

 

 

 

 

 

 

NA4

NA4

 

 

 

 

 

 

 

 

 

 

NA5

NA5

 

 

 

 

 

 

 

 

 

 

NA6

NA6

 

 

 

 

 

 

 

 

 

 

NA7

NA7

 

 

 

 

 

 

 

 

 

 

Note:NA=NAStandards,BL=BlankControl,TS=TestSamples.

 

Table2Reagentcompositionforeachwell

NAStandard

BlankControl

TestSample

SerialDilutions*:50μL

AssayBuffer:50μL

50μL

*Note:Addtheserialdilutionsofneuraminidasestandardfrom0.312mUto20mUintowellsfromNA1toNA7induplicate.

 

5.Runneuraminidaseassay:

5.1   Add50μLofneuraminidaseassaymixture(fromStep3)toeachwelloftheneuraminidasestandard,blankcontrol,andtestsamples(seeStep4.3)tomakethetotalneuraminidaseassayvolumeof100µL/well.

Note:Fora384-wellplate,add25μLofsampleand25μLofneuraminidasereactionmixtureintoeachwell.

 

5.2   Incubatethereactionat37oCorroomtemperaturefor1to2hours,protectedfromlight.

Note:37oCincubationgivesbetterresults.

 

5.3   MonitorthefluorescenceincreaseatEx/Em=320/460nm(cutoff=420nm)withafluorescencemicroplatereader.

References&Citations
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  1. SandbulteMR,GaugerPC,KitikoonP,ChenH,PerezDR,RothJA,VincentAL.(2016)NeuraminidaseinhibitingantibodyresponsesinpigsdifferbetweeninfluenzaAvirusN2lineagesandbyvaccinetype.Vaccine,34,3773.
  2. ZhangM,ZhaoH,ZhaoZ,YanH,LvR,CuiL,YuanJ,WangD,GengY,LiuD,WangX.(2016)Rapidscreening,identification,andpurificationofneuraminidaseinhibitorsfromLithospermumerythrorhizonSieb.etZucc.byultrafiltrationwithHPLC-ESI-TOF-MScombinedwithsemipreparativeHPLC.JSepSci,39,2097.
  3. HuangW,TanM,ZhaoX,ChengY,LiX,GuoJ,WeiH,XiaoN,WangZ,WangD,ShuY.(2015)[SusceptibilityofhumaninfluenzaA(H3N2)virusestoneuraminidaseinhibitorsisolatedduring2011-2012inChina].ZhonghuaYuFangYiXueZaZhi,49,481.
  4. SomasundaramB,FeeCJ,FredericksR,WatsonAJ,FairbanksAJ.(2015)Developmentofasurfaceplasmonresonanceassaytomeasurethebindingaffinityofwild-typeinfluenzaneuraminidaseanditsH274Ymutanttotheantiviraldrugzanamivir.JMolRecognit,28,87.
  5. PedersenJC.(2014)Neuraminidase-inhibitionassayfortheidentificationofinfluenzaAvirusneuraminidasevirussubtypeorneuraminidaseantibodyspecificity.MethodsMolBiol,1161,27.
  6. SmolonoginaTA,DeshevaIuA,RekstinAR,MironovAN,RudenkoLG.(2013)[Evaluationoftheanti-neuraminidaseantibodiesinclinicaltrialsoftheliveinfluenzavaccineoftheA(H5N2)subtype].VoprVirusol,58,31.
  7. LuX,LiuF,ZengH,SheuT,AchenbachJE,VeguillaV,GubarevaLV,GartenR,SmithC,YangH,StevensJ,XuX,KatzJM,TumpeyTM.(2014)Evaluationoftheantigenicrelatednessandcross-protectiveimmunityoftheneuraminidasebetweenhumaninfluenzaA(H1N1)virusandhighlypathogenicavianinfluenzaA(H5N1)virus.Virology,454-455,169.
  8. ZhangY,FuD,ChenH,ZhangZ,ShiQ,ElsayedAK,LiB.(2013)PartialantiviralactivitiesdetectionofchickenMxjointingwithneuraminidasegene(NA)againstNewcastlediseasevirus.PLoSOne,8,e71688.
  9. ChoiKS,KyeSJ,JeonWJ,ParkMJ,KimS,SeulHJ,KwonJH.(2013)Preparationanddiagnosticutilityofahemagglutinationinhibitiontestantigenderivedfromthebaculovirus-expressedhemagglutinin-neuraminidaseproteingeneofNewcastlediseasevirus.JVetSci,14,291.
  10. HurtAC,Okomo-AdhiamboM,GubarevaLV.(2012)Thefluorescenceneuraminidaseinhibitionassay:afunctionalmethodfordetectionofinfluenzavirusresistancetotheneuraminidaseinhibitors.MethodsMolBiol,865,115.

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