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AntibodiesOnline/Nitric Oxide Colorimetric Detection Kit/ABIN577678/2 x 96 tests188bio精品生物—专注于实验室精品爆款的电商平台 - 蚂蚁淘旗下精选188款生物医学科研用品
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AntibodiesOnline/Nitric Oxide Colorimetric Detection Kit/ABIN577678/2 x 96 tests
Antigen
NitricOxide(NO)
MinimumDetectionLimit
3.0μM
Application
BiochemicalAssay(BCA)
Options
Bulkdiscount
Supplier
SupplierProductNo.
PurposeTheDetectX®NitricOxideDetectionKitisdesignedtoquantitativelymeasureNitrateandNitritepresentinavarietyofsamples.NitricOxidecontentisderivedfromthesumofNitrate(-NO3)andNitrite(-NO2).
BrandDetectX®
SampleTypeWater,BIOLOGicalBuffers,Serum,Plasma,Urine,Saliva,TissueCultureSupernatant
DetectionMethodColorimetric
SpecificitySpeciesIndependent.SamplesTypesvalidated:Water,Buffers,Serum,Plasma,Urine,SalivaandTCM
Sensitivity2.63µMintheNitriteand1.02µMintheTotalNitricOxideassays.
CharacteristicsTheNitricOxide(NO)DetectionKitisdesignedtoquantitativelymeasureNitrateandNitritepresentinavarietyofsamples.NitricOxidecontentisderivedfromthesumofNitrateandNitrite.BothNitrateandNitritestandardsareprovidedtogeneratestandardcurvesfortheassayandallsamplesshouldbereadofftheappropriatestandardcurve.ForNitriteDetection,samplesaremixedwiththeColorReagentsAandBandincubatedatroomtemperaturefor5minutes.Thecoloredproductisreadat550-570nm.TotalNitricOxidecontentismeasuredafterthesampleisincubatedwithNitrateReductaseandNADH.ThereductaseincombinationwithNADHreducesNitratetoNitrite.Aftera20minutein-cubationatroomtemperature,ColorReagentsAandBareaddedandincubatedatroomtempera-turefor5minutes.TheconcentrationofNitrateinthesampleiscalculatedbytakingthemeasuredNitriteconcentrationandsubtractingfromtheTotalNitricOxideconcentrationforthesample.
ComponentsClear96wellPlates2Plates
NitrateStandard200μLSodiumNitrateat2,000μMinaspecialstABIlizingsolution.CalibratedtoNISTStandardReferenceMaterialLotNumber3185NitriteStandard200μL
SodiumNitriteat2,000μMinaspecialstabilizingsolution.CalibratedtoISO/IEC17025
AssayBuffer60mLAbuffercontainingdetergentsandstabilizers.
NADHConcentrate1.2mLReducedß-nicotinamideadeninedinucleotide(NADH)asastablesolution.
NitrateReductase1VialNitrateReductase(NR)asastablesolidstoredinadesiccator.
EnzymeStabilizationBuffer1mLAbuffercontainingspecialstabilizersforNR.
ColorReagentA5mLAsolutionofSulfanilamideinacid.CAUTION:CAUSTIC
ColorReagentB5mLAsolutionofN-(1-Naphthyl)ethylenediamineinacid.CAUTION:CAUSTIC
MaterialnotincludedDistilledordeionizedwaterfreeofdetectablenitrateornitrite.10,000MolecularWeightCutOff(MWCO)polysulfonefilters(CorningSpin-XUF500,431478)orsimilarproduct.
RepeaterpipetsusingdisposabletipsforadditionofColorReagentsA&B,NADHandNitrateReductase.96wellplatereadercapableofreADIngopticalabsorptionat540-570nm.
Softwareforconvertingopticaldensity(OD)readingsfromtheplatereaderandcarryingoutfourparameterlogisticcurve(4PLC)fitting.
AlternativeNameNitricOxide
BackgroundNitricoxide(NO)isadiffusIBLe,transient,reactivemoleculethathasphysiologicaleffectsinthepicomolar-to-micromolarrange.Actingthroughsolubleguanylatecyclaseactivation,NOisanimportantphysiologicalregulatorofthecardiovascular,nervous,andimmunologicalsystems1.NOisbio-availablebytworoutes.Itcanbeendogenouslygeneratedbyconstitutiveorinduceden-zymeslikeNitricOxideSynthaseoritcanbeorallyingestedasnitrates/nitritesforrapiduptakeintocirculationandsubsequentconversion2.Thereactivenatureofnitricoxideallowsittoactasacytotoxicfactorwhenreleasedduringanimmuneresponsebycellssuchasmacrophages.ThereactivityalsoallowsNOtobeeasilycon-vertedtoatoxicradicalthatcanproducenitrosativedamagetocells,organellesandmoleculessuchasDNA.Nitrosaylationhowevercanbearegulatedpost-translationalmodificationincellsignaling3.Thebalanceanddynamicsoftheregulatory/damagefacetsofNOaremajorforcesinmitochondrialsignalinganddysfunction4.NOislinkednotonlytocoronaryheartdisease,en-dothelialdysfunctions,erectiledysfunction,andneurologicaldisorders,butalsodiabetes,chronicperiodontitis,autism,cancer,andassortedage-relateddiseases5-9.ThephysicalpropertiesofNitricOxidemakeitchallengingfordirectdetectionmethods.How-ever,colorimetricmethodscanbeappliedtomeasureitsstablebreak-downproductsnitrate(-NO)andnitrite(-103NO2)
ApplicationNotesNO,NitrateandNitriteisidenticalacrossspeciesandthiskitwillmeasureNOfromallsources.
WedeterminedNOinhumansamplesandtheendusershouldevaluaterecoveriesofNOinsamplesfromotherspeciesbeingtested.
ThekitwillmeasureNOincellculturemedium,howevermanymediacontainnitratesalts.
CareneedstobetakenintheselectionofmediawhenNOmeasure-mentistobedone.
Ifsamplesneedtobestoredaftercollection,werecommendstoringthemat-70 °Corlower,pref-erablyafterbeingfrozeninliquidnitrogen.
Thisassayhasbeenvalidatedforserum,plasma,urine,andsaliva,aswellaswaterandbuffersamples.
Tris,HEPES,andPBSbuffersarecompatibleat pH7.2,asisEDTAat≤10mM.
DetergentssuchasTritonX-100,Tween20andCHAPSarecompatibleatconcentrationsof≤0.1 %.
Mostcelllysatesandtissuehomogenatesshouldalsobecompatible.
Samplescontainingthesedetergentsshouldbedilutedatleast1:2withtheAssayBuffer.
SamplescontainingSDSorazidearenotcompatiblewiththeassay.
Samplescontainingvisibleparticu-lateshouldbecentrifugedpriortofiltrationandusing.
AssayTime0.5h
Plate96wells
ProtocolBothNitrateandNitritestandardsareprovidedtogeneratestandardcurvesfortheassayandallsamplesshouldbereadofftheappropriatestandardcurve.
ForNitritedetection,samplesaremixedwiththeColorReagentsAandBandincubatedatroomtemperaturefor5minutes.
Thecoloredproductisreadat550-570nm.
TheconcentrationofNitriteinthesampleiscalculated,aftermakingasuitablecorrectionforanydilutionofthesample,usingsoftwareavailablewithmostplatereaders.
TotalNitricOxidecontentismeasuredafterthesampleisincubatedwithNitrateReductaseandNADH.
ThereductaseincombinationwithNADHreducesNitratetoNitrite.
Aftera20minuteincubationatroomtemperature,ColorReagentsAandBareaddedandincubatedatroomtem-peraturefor5minutes.
ThecoloredproductisreadandcalculatedaswiththeNitritedetermina-tionabove.
TheconcentrationofNitrateinthesampleiscalculatedbysubtractingthemeasuredNitriteconcentrationfromtheTotalNitricOxideconcentrationforthesample.
ThiskitusesNitrateandNitriteStandardsolutionscalibratedtotheUSNationalInstituteforSci-enceandTechnologyStandardReferenceMaterialsandISO/IECstandards.
ReagentPreparation

Allowthekitreagentstocometoroomtemperaturefor30minutes.
WerecommendthatallstandardsandsamplesberuninduplicatetoallowtheendusertoaccuratelydetermineNOcon-centrations.
Ensurethatallsampleshavereachedroomtemperatureandhavebeendilutedandfilteredthrougha10,000MWCOfilterpriortorunningtheminthekit.
NitrateReductase(NR)Allowthedesiccatortowarmtoroomtemperature.
Add550μLofEnzymeStabilizationBuffertothevial.
Vortexgentlyandallowtositatroomtemperaturefor5minutes.
Forextendedperiodsoftime(>2hours)storereconstitutedNRonice.
StoreanyunusedreconstitutedNRat-20°C.
PrepareNRforuseintheassaybytakingonepartofreconstitutedNRandaddingtothreepartsofAssayBuffer.
SeeTablebelow.
NitrateReductaseDilutionTable1/2PlateOnePlateTwoPlatesReconstitutedNR150μL275μL500μLAssayBuffer450μL825μL1.5mLTotalVolume600μL1.1mL2mL®Forextendedperiodsoftime(>2hours)storereconstitutedNRonice.www.ArborAssays.com7WEBINSERT150513reagentpreparatiOncOntinuedNADHPreparationPrepareNADHbydilutingonepartofNADHConcentratewithanequalpartofAssayBuffer.
NADHDilutionTable1/2PlateOnePlateTwoPlatesNADHConcentrate300μL550μL1mLAssayBuffer300μL550μL1mLTotalReactionMixVolume600μL1.1mL2mLDonotstoredilutedNADH.
StandardPreparationNitrateandNitriteStandardsarepreparedidenticallybylabelingseventesttubesas#1through#7.
Brieflyvortextomixandthenspinthevialofstandardinamicrocentrifugetoensurecontentsareatbottomofvial.
Pipet360μLofAssayBufferintotube#1and200μLintotubes#2to#7.
Care-fullyadd40μLofeitherthe-NO2or-NO3Standardtotube#1andvortexcompletely.
Take200μLofthesolutionintube#1andaddittotube#2andvortexcompletely.
Repeatthisfortubes#3through#7.
TheconcentrationofNitrateorNitriteintubes1through7willbe200,100,50,25,12.5,6.25and3.125μM.
UseallStandardswithin2hoursofpreparation.

SamplePreparation

Allsamplesmustbefilteredthrougha10,000MWCOspinfiltertoremoveprotein.Serum,plasma,saliva,orurineDilutesamplewithAssayBufferandfilterthrougha10,000MWCOdevicefollowingthemanu-facturersrecommendations.CollectthefiltratesandeitherfurtherdilutewithAssayBufferasappropriateorusedirectlyintheassay.Forserumandplasma,therecommendedfinaldilutionis≥1:4.Forurineandsaliva,therecommendedfinaldilutionis≥1:8.

AssayProcedure

UsetheappropriatestandardsforeitherNitrite(-NO2)orNitrate(-NO3)determination.Allsam-plesshouldbedilutedandfilteredthrougha10,000MWCOfilterpriortousing.NitriteDeterminationProtocol
1.Usetheplatelayoutsheetonthebackpagetoaidinpropersampleandstandardidentification.
2.Pipet50μLofsamplesorNitritestandardsintoduplicatewellsintheplate.
3.Pipet50μLofAssayBufferintoduplicatewellsastheZerostandard.
4.Add25μLoftheColorReagentAtoeachwellusingarepeaterpipet.
5.Add25μLoftheColorReagentBtoeachofwellusingarepeaterpipet.
6.Incubateatroomtemperaturefor5 minutes.
7.Readtheopticaldensityat540-570nm.ThesereadingsarefortheNitritedetermination.TotalNitricOxideDeterminationProtocol
1.Usetheplatelayoutsheetonthebackpagetoaidinpropersampleandstandardidentification.
2.Pipet50μLofsamplesorNitratestandardsintoduplicatewellsintheplate.
3.Pipet50μLofAssayBufferintoduplicatewellsastheZerostandard.
4.Add10μLofpreparedNADHtoeachwellusingarepeaterpipet.
5.Add10μLofpreparedNRtoeachwellusingarepeaterpipet.
6.Incubateatroomtemperaturefor20 minutes.
7.Add25μLoftheColorReagentAtoeachwellusingarepeaterpipet.
8.Add25μLoftheColorReagentBtoeachofwellusingarepeaterpipet.
9.Incubateatroomtemperaturefor5 minutes.10.Readtheopticaldensityat540-570nm.ThesereadingsarefortheTotalNitricOxidedetermination.

CalculationofResults

Averagetheduplicateopticaldensityreadingsforeachstandardandsample.
Createastandardcurvebyreducingthedatausingthe4PLCfittingroutineontheplatereader,aftersubtractingthemeanODsforthezerostandard.
Theconcentrationsobtainedshouldbemultipliedbythedilutionfactortoobtainsamplevalues.
Or,usetheonlinetoolfromhttp://www.myassays.com/arbor-assays-nitric-oxide-colorimetric-kit.assaytocalculatethedata.
QRcodeforDataAnalysis:*TheMyAssayslogoisaregisteredtrademarkofMyAssaysLtd.
Nitrite(-NO2)concentrationsarecalculatedfromthedataobtainedfromtheNitriteProtocolstan-dardcurvedatautilizingthecurvefittingroutinesuppliedwiththeplatereader.
TotalNOconcentrationsarecalculatedfromthedataobtainedfromtheTotalNitricOxideProto-col(nitrite+nitrate)standardcurvedatautilizingthecurvefittingroutinesuppliedwiththeplatereader.
Nitrate(-NO3)concentrationsareobtainedbysubtractingthe-NO2concentrationsofeachsamplefromtheTotalNOconcentrations.
SeeBelow:Nitrate(-NO3)=TotalNO-Nitrite(-NO2)

RestrictionsForResearchUseonly
PrecautionofUseAswithallsuchproducts,thiskitshouldonlybeusedbyqualifiedpersonnelwhohavehadlabo-ratorysafetyinstruction.
Thecompleteinsertshouldbereadandunderstoodbeforeattemptingtousetheproduct.
TheColorReagentsAandBarebothacidsolutionsandshouldbehandledlikeanylaboratoryacid.
Storage-20°C,4°C,RT
StorageCommentAllcomponentsofthiskitshouldbestoredat4°Cuntiltheexpirationdateofthekit.Oncereconstituted,theNitrateReductasemustbestoredat-20°C.
SupplierImages
image for Nitric Oxide Colorimetric Detection Kit (ABIN577678)NitricOxideColorimetricDetectionKit
Productcitedin:Carnevale,Loffredo,Nocella,Bartimoccia,Sanguigni,Soresina,Plebani,Azzari,Martire,Pignata,Violi:"Impairedplateletactivationinpatientswithhereditarydeficiencyofp47(phox)."in:Britishjournalofhaematology,2016(PubMed).

Costarelli,Giacconi,Malavolta,Basso,Piacenza,Provinciali,Maggio,Corsonello,Lattanzio:"Differenttranscriptionalprofilingbetweensenescentandnon-senescenthumancoronaryarteryendothelialcells(HCAECs)byOmeprazoleandLansoprazoletreatment."in:Biogerontology,2016(PubMed).

Vozzi,Lucarini,Dicarlo,Andreoni,Salvolini,Ferretti,Mattioli-Belmonte:"InvitroLifespanandsenescentbehaviourofhumanperiostealderivedstemcells."in:Bone,Vol.88,

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