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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 Peroxynitrite Quantification Kit *Green Fluorescence*/16316/100 Tests
产品编号:16316
市  场 价:¥100060.00
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:$5003.00
品      牌: AAT Bioquest
公      司:AAT Bioquest
公司分类:
AAT Bioquest/Amplite™ Fluorimetric Peroxynitrite Quantification Kit *Green Fluorescence*/16316/100 Tests
商品介绍
Overview
PrinterFriendlyVersion

Ex/Em(nm)502/535
MWN/A
CAS#N/A
SolventN/A
StorageF/D/L
CategoryNeuroBIOLOGy
ReactiveOxygenSpecies
RelatedCellSignaling
BiochemicalAssays
Peroxynitrite(ONOO-)isastrongoxidizingspeciesandahighlyactivenitratingagent.PeroxynitriteisformedfromthereactionbetweensuperoxiderADIcalsandnitricoxidegeneratedincells.Itcandamageawidearrayofbiomoleculesincludingproteins,enzymes,lipidsandnucleicacids,eventuallycontributingtocelldeath.Duetoitsextremelyshorthalf-lifeandlowsteady-stateconcentration,ithasbeenchallengingtodetectandquantifyperoxynitriteinsolution.Inordertoaddressthisneed,AATBioquest"sAmplite™FluorimetricPeroxynitriteQuantificationKitprovidesasensitivetooltomeasureONOO-insolution.DAX-J2™PONGreen99reactswithONOO-togenerateabrightgreenfluorescentproduct.ItspecificallyreactswithONOO-withhighselectivityoverotherreactiveoxygenspecies(ROS)andreactivenitrogenspecies(RNS).Thiskitcanbeusedwithafluorescencemicroplatereaderandspectrometer.
SpectrumAdvancedSpectrumViewer



Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.Prepare500XDAX-J2™PONGreen99stocksolution:

Add20µLofDMSO(ComponentC)intothevialofDAX-J2™PONGreen99(ComponentA)tomake500Xstocksolution.

Note:20μLofreconstitutedDAX-J2™PONGreen99stocksolutionisenoughfor1plate.Unusedportioncanbealiquotedandstoredat≤-20°Cformorethanonemonthifthetubesaresealedtightlyandkeptfromlight.Avoidrepeatedfreeze-thawcycles.

2.PrepareONOO¯assaymixture:

Add20µLof500XDAX-J2™PONGreen99stocksolution(fromStep1)into10mLofAssayBuffer(ComponentB),andmixthemwell.

Note:Thisassaymixtureisenoughforone96-wellplate.Itisstableat4ºCfor2hourswhenprotectedfromlight.

3.PrepareONOO¯stocksolution(notprovided):

Peroxynitritestocksolutionwassynthesizedaccordingtoliteraturereport.Briefly,amixtureofsodiumnitrite(0.6M)andhydrogenperoxide(0.7M)wasacidifiedwithhydrochloricacid(0.6M),andsodiumhydroxide(1.5M)wasaddedwithin1-2secondstomakethesolutionalkaline.Theexcesshydrogenperoxidewasremovedbypassingthesolutionthroughashortcolumnofmanganesedioxide.Theextinctioncoefficientofperoxynitritesolutionin0.1MNaOHis1670M-1cm-1at302nm.TheONOO¯stocksolutionisnotstable;youmightstoreitat≤-80°Cfor2-4weeks,wehighlyrecommendmakeitfreshtouse.

4.PrepareserialdilutionsofONOO¯standard(0to20µM):

4.1   DiluteONOO¯stocksolution(fromStep3)inAssaybuffer(ComponentB)tohave20µMONOO¯standardsolution,andthenperform1:2serialdilutionstogetapproximately20,10,5,2.5,1.25,0.625,0.313and0µM.

4.2   AddONOO¯standardsandONOO¯containingtestsamplesintoa96-wellsolidblackmicroplateasdescribedinTables1and2.

 

Table1.LayoutofONOO¯standardsandtestsamplesinasolidblack96-wellmicroplate

BL

BL

TS

TS

….

….

 

 

 

 

 

 

O1

O1

….

….

….

….

 

 

 

 

 

 

O2

O2

 

 

 

 

 

 

 

 

 

 

O3

O3

 

 

 

 

 

 

 

 

 

 

O4

O4

 

 

 

 

 

 

 

 

 

 

O5

O5

 

 

 

 

 

 

 

 

 

 

O6

O6

 

 

 

 

 

 

 

 

 

 

O7

O7

 

 

 

 

 

 

 

 

 

 

       *Note:O=ONOO¯Standards;BL=BlankControl;TS=TestSamples

 

Table2.Reagentcompositionforeachwell

ONOO¯Standard

BlankControl

TestSample

Serialdilutions*(50μL)

TE:50μL

50μL

*Note:AddtheseriallydilutionsofONOO¯standardsfrom0.313to20µmintowellsfromO1toO7induplicate.

 

5.RunONOO¯assay:

5.1   Add50μLofassaymixture(fromStep2)toeachwelloftheONOO¯standard,blankcontrol,andtestsamples(seeStep4.2)tomakethetotalassayvolumeof100µL/well.

Note:Fora384-wellplate,add25μLsampleand25μLofassaymixtureperwell.

5.2   Incubatethereactionatroomtemperaturefor5to10minutes,protectedfromlight.

5.3   MonitorthefluorescenceincreaseatEx/Em=490/530nm(cutoffat515nm)withafluorescenceplatereader.

References&Citations
CitationExplorer

Fluorescentreal-timequantitativemeasurementsofintracellularperoxynitritegenerationandinhibition
Authors:ZhenLuo,QinZhao,JixiangLiu,JinfangLiao,RuoguPeng,YuntingXi,ZhenjunDiwu
Journal:Analyticalbiochemistry(2017):44--48

GenesilencingofendothelialvonWillebrandFactorattenuatesangiotensinII-inducedendothelin-1expressioninporcineaorticendothelialcells
Authors:AnarDushpanova,SilviaAgostini,EnricaCiofini,ManuelaCABIati,ValentinaCasieri,MarcoMatteucci,SilviaDelRy,AldoClerico,SergioBerti,VincenzoLionetti
Journal:ScientificReports(2016)


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