| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 571/585 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | EnzymeDetection RedoxEnzymes |
| Related | MicroplateReaders BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1.Preparestocksolutions:
1.1 250XAmplite™HRPSubstratestocksolution:Add100µLofDMSO(ComponentD)intothevialofAmplite™HRPSubstrate(ComponentA).Thestocksolutionshouldbeusedpromptly;anyunusedsolutionshouldbealiquotedandrefrozenat-20oC.Avoidrepeatedfreeze-thawcycles.
Note1:Amplite™HRPSubstrateisunstableinthepresenceofthiolssuchasDTT,glutathione(reducedform:GSH)andβ-mercaptoethanol.Thepresenceofthiolsatconcentrationhigherthan10μMwouldsignificantlydecreasetheassaydynamicrange.
Note2:Somedetergents(suchasBrij-35,Tween-20andNP40),NADHandNADPHalsointerferewiththeassay.
1.2 50U/mLHorseradishPeroxidasestocksolution:Add1mLofAssayBuffer(ComponentB)intothevialofHorseradishPeroxidase(ComponentC).Note:TheunusedHRPsolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.
2.Prepareassayreactionmixture:
Prepareassayreactionmixtureaccordingtothefollowingtablesandkeepfromlight.
Table1.2XAssayreactionmixtureforone96-wellplate
Components | Volume |
Amplite™HRPSubstratestocksolution(250X,fromStep1.1) | 20μL |
50U/mLHorseradishPeroxidase(fromStep1.2) | 20μL |
AssayBuffer(ComponentB) | 5mL |
Totalvolume | 5mL |
Table2.LayoutofLysylOxidasestandardsandtestsamplesinasolidblack96-wellmicroplate
BL | BL | TS | TS | …. | …. |
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LS1 | LS1 | …. | …. | …. | …. |
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LS2 | LS2 |
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LS3 | LS3 |
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LS4 | LS4 |
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LS5 | LS5 |
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LS6 | LS6 |
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LS7 | LS7 |
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Note:LS=LysylOxidaseStandards,BL=BlankControl,TS=TestSamples.
Table3.Reagentcompositionforeachwell
LysylOxidaseStandards | BlankControl | TestSample |
SerialDilutions*:50μL | AssayBuffer(ComponentB):50μL | 50μL |
*Note1:AddtheseriallydilutedLysylOxidasestandardsfrom0.04to4μg/mLintowellsfromLS1toLS7induplicate.
Note2:HighconcentrationofLysylOxidasemaycausereducedfluorescencesignalduetotheoveroxidationofAmplite™HRPSubstrate(toanon-fluorescentproduct).
Note3:LysylOxidasestandardsareforpositivecontrolonly,andshouldnotbereliedonasaquantitationstandardforenzymeactivity.
3.Runlysyloxidaseassayinsupernatants:
3.1 Add50μLofassayreactionmixture(fromStep2)intoeachwelloflysyloxidasestandard,blankcontrol,andtestsamples(seeStep2,Table3)tomakethetotallysyloxidaseassayvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLofassayreactionmixtureintoeachwell.
3.2 Incubatethereactionat37oCfor10to30minutes,protectedfromlight.
3.3 MonitorthefluorescenceincreasewithafluorescenceplatereaderatEx/Em=540/590nm.
Note:Thecontentsoftheplatecanalsobetransferredtoawhiteclearbottomplateandreadbyanabsorbancemicroplatereaderatthewavelengthof576±5nm.Theabsorptiondetectionhaslowersensitivitycomparedtofluorescencereading.
4.Runlysyloxidaseassayforcells:
TheAmplite™FluorimetricLysylOxidaseAssayKitcanbeusedtomeasurethereleaseofactivelysyloxidasefromcells.Thefollowingisasuggestedprotocolthatcanbemodifiedaccordingtoyourspecificresearchneeds.
4.1 Preparecellsina96-wellplate(50-100μL/well),andactivatethecellsasdesired.Harvestthecellmedia.
Note:Thenegativecontrols(mediaaloneandnon-activatedcells)areincludedformeasuringbackgroundfluorescence.
4.2 Add50μLofassayreactionmixture(fromStep2)intoeachwellofthecellmedia(fromStep4.1),andthoseoflysyloxydasestandards(fromStep2).
Note:Fora384-wellplate,add25μLofcellmediaand25μLofassayreactionmixtureintoeachwell.
4.3 Incubatethereactionat37oCfor10to30minutes,protectedfromlight.
4.4 MonitorthefluorescenceincreasewithafluorescenceplatereaderatEx/Em=530to570/590to600nm(maximumEx/Em=540/590nm,cutoff570nm).
| References&Citations |  CitationExplorer |
Detrimentalroleoflysyloxidaseincardiacremodeling
Authors:EliaCElHajj,MiladCElHajj,VanKNinh,JessicaMBradley,MarioAClaudino,JasonDGardner
Journal:JournalofMolecularandCellularCardiology(2017)
EndothelialAntioxidant-1:aKeyMediatorofCopper-dependentWoundHealinginvivo
Authors:ArchitaDas,VaradarajanSudhahar,Gin-FuChen,HaWonKim,Seock-WonYoun,LydiaFinney,StefanVogt,JayYang,JunghunKweon,BayasgalanSurenkhuu
Journal:ScientificReports(2016)
Inhibitionoflysyloxidasebycortisolregenerationinhumanamnion:Implicationsforruptureoffetalmembranes
Authors:ChaoLiu,ChunmingGuo,WangshengWang,PingZhu,WenjiaoLi,YabingMi,LeslieMyatt,KangSun
Journal:Endocrinology(2016):4055--4065
InhibitionoflysyloxidasebyprostaglandinE2viaEP2/EP4receptorsinhumanamnionfibroblasts:Implicationsforparturition
Authors:ChaoLiu,PingZhu,WangshengWang,WenjiaoLi,QunShu,Zi-JiangChen,LeslieMyatt,KangSun
Journal:Molecularandcellularendocrinology(2016):118--127
LOXFailstoSubstituteforRANKLinOsteoclastogenesis
Authors:MasayukiTsukasaki,KokiHamada,KazuoOkamoto,KazukiNagashima,AsukaTerashima,NorikoKomatsu,StephanieJWin,TadashiOkamura,TakeshiNitta,HisatakaYasuda
Journal:JournalofBoneandMineralResearch(2016)
ReversalofVascularCalcificationandAneurysmsinaRatModelUsingDualTargetedTherapywithEDTA-andPGG-LoadedNanoparticles
Authors:NasimNosoudi,AniqaChowdhury,StevenSiclari,SakethKaramched,VaideeshParasaram,JoeParrish,PatrickGerard,NarendraVyavahare
Journal:Theranostics(2016):1975
SuppressionofPhosphatidylinositol3-Kinase/AktSignalingAttenuatesHypoxia-InducedPulmonaryHypertensionThroughtheDownregulationofLysylOxidase
Authors:Xiao-DongXia,JasmineLee,SajidKhan,LepingYe,YuanLi,LiangDong
Journal:DNAandCellBiology(2016):599--606
Attenuationoflipopolysaccharide-inducedlungvascularstiffeningbylipoxinreduceslunginflammation
Authors:FanyongMeng,IsaMambetsariev,YufengTian,YvonneBeckham,AngeloMeliton,AlanLeff,MargaretLGardel,MichaelJAllen,KonstantinGBirukov,AnnaABirukova
Journal:Americanjournalofrespiratorycellandmolecularbiology(2015):152--161
Attenuationoflysyloxidaseandcollagengeneexpressioninkeratoconuspatientcornealepitheliumcorrespondstodiseaseseverity
Authors:RohitShetty,ArunapriyaSathyanarayanamoorthy,ReshmaAirodyRamachandra,VishalArora,AnupritaGhosh,PurnimaRamanSrivatsa,NatashaPahuja,RudyMMANuijts,AbhijitSinha-Roy,RajivRMohan
Journal:(2015)
Coppertransportproteinantioxidant-1promotesinflammatoryneovascularizationviachaperoneandtranscriptionfactorfunction
Authors:Gin-FuChen,VaradarajanSudhahar,Seock-WonYoun,ArchitaDas,JaehyungCho,TetsuroKamiya,NorifumiUrao,RonaldDMcKinney,BayasgalanSurenkhuu,TakaoHamakubo
Journal:Scientificreports(2015)
