| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 571/585 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | EnzymeDetection Phosphodiesterases |
| Related | BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1.PrepareSphingomyelinase(SMase)workingsolution:
1.1 Prepare100XSMasestocksolutionbyadding50µLofPBSwith0.1%BSAintothevialofSphingomyelinase(ComponentB).
Note:Theunused100XSMasestocksolutionshouldbealiquotedandstoredat-20oC
1.2 PrepareSMaseworkingsolutionbyaddingthewholecontent(50µL)of100XSMasestocksolution(fromStep1.1)into5mLofSMaseReactionBuffer(ComponentD),andmixwell.
Note:TheSMaseworkingsolutionshouldbeusedpromptly.
2.Preparesphingomyelinstandardsand/orsphingomyelinase-containingsamples:
2.1 Add2µLof50mMSphingomyelin(ComponentF)into1000µLofSMaseReactionBuffer(ComponentD)togeta100µMSphingomyelinstandardsolution.
2.2 Take200μLof100µMSphingomyelinstandardsolutiontoperform1:3serialdilutionstoget30,10,3,1,0.3,0.1and0µMseriallydilutedsphingomyelinstandards.
2.3 Addthesphingomyelinstandardsandsphingomyelin-containingtestsamplesintoasolidblack96-wellmicroplateasshowninTables1and2.
Note:Treatyourcellsortissuesamplesasdesired.
Table1Layoutofsphingomyelinstandardsandtestsamplesinasolidblack96-wellmicroplate
BL | BL | TS | TS | …. | …. |
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SM1 | SM1 | …. | …. | …. | …. |
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SM2 | SM2 |
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SM3 | SM3 |
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SM4 | SM4 |
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SM5 | SM5 |
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SM6 | SM6 |
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SM7 | SM7 |
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Note:SM=SphingomyelinStandards,BL=BlankControl,TS=TestSamples.
Table2Reagentcompositionforeachwell
SphingomyelinStandards | BlankControl | TestSample |
SerialDilutions:50μL | SMaseReactionBuffer:50μL | 50μL |
Note:Addtheseriallydilutedsphingomyelinstandardsfrom0.1to100µMintowellsfromSM1toSM7induplicate.
2.4 Add50μLofSMaseworkingsolution(fromStep1.2)intoeachwellofsphingomyelinstandards,blankcontrolandtestsamples(fromStep2.3).
2.5 Incubatethereactionmixtureat37°Cfor1-2hours.
3.Prepare200XAmplite™Redstocksolution:
Add80µLofDMSO(ComponentG)intothevialofAmplite™Red(ComponentC)tomake200XAmplite™Redstocksolution.
Note1:TheunusedAmplite™Redstocksolutionshouldbealiquotedandstoredat-20oC(keptfromlight).
Note2:TheAmplite™Redisunstableinthepresenceofthiols(suchasDTTand2-mercaptoethanol).ThefinalconcentrationofDTTor2-mercaptoethanolinthereactionshouldbelowerthan10μM.Amplite™RedisalsounstableathighpH(>8.5).ThereactionsshouldbeperformedatpH7-8.pH7.4isrecommendedfortheassaybuffer.
4.Preparesphingomyelinassaymixture:
4.1 Addthewholecontent(5mL)ofAssayBuffer(ComponentE)intothebottleofEnzymeMix(ComponentA),andmixthemwell.
4.2 Add25µL200XAmplite™Redstocksolution(fromStep3)intothebottleofEnzymeMixsolution(fromStep4.1)tomakethesphingomyelinassaymixturebeforestartingtheassay.
Note:Thesphingomyelinassaymixtureshouldbeusedpromptlyandkeptfromlight;longerstorageislikelytocausehighassaybackground.
5.Runsphingomyelinassay:
5.1 Add50μLofsphingomyelinassaymixture(fromStep4.2)intoeachwellofsphingomyelinstandards,blankcontrol,andtestsamples(fromStep2.5)tomakethetotalsphingomyelinaseassayvolumeof150µL/well.
Note:Fora384-wellplate,add25μLofsample,25μLofsphingomyeliaseworkingsolutionand25μLofsphingomyelinassaymixtureintoeachwell.
5.2 Incubatethereactionmixturefor1-2hoursatroomtemperature(protectedfromlight).
5.3 MonitorthefluorescenceincreasewithafluorescencemicroplatereaderatEx/Em=540/590nm(cutoffat570nm).
| References&Citations |  CitationExplorer |
Doxepinmitigatesnoiseinducedneuronaldamageinprimaryauditorycortexofmiceviasuppressionofacidsphingomyelinase/ceramidepathway
Authors:Yu-TingSu,Xing-XingMeng,XiZhang,Yi-BinGuo,Hai-JunZhang,Yao-PingCheng,Xiao-PingXie,Yao-MingChang,Jun-XiangBao
Journal:TheAnatomicalRecord(2017)
NewAspectsofSilibininStereoisomersandtheir3-O-galloylDerivativesonCytotoxicityandCeramideMetabolisminHepG2hepatocarcinomaCellLine
Authors:MahdiMashhADIAkbarBoojar,ShahramEjtemaeiMehr,MahsaHassanipour,MasoudMashhadiAkbarBoojar,AhmadRezaDehpour
Journal:IranianJournalofPharmaceuticalResearch(2016):421--433
RiccardinDNinduceslysosomalmembranepermeABIlizationbyinhibitingacidsphingomyelinaseandinterferingwithsphingomyelinmetabolisminvivo
Authors:LinLi,HuanminNiu,BinSun,YananXiao,WeiLi,HuiqingYuan,HongxiangLou
Journal:ToxicologyandAppliedPharmacology(2016):175--184
MitochondrialrespirationcontrolslysosomalfunctionduringinflammatoryTcellresponses
Authors:FrancescBaixauli,RebecaAcín-Pérez,CarolinaVillarroya-Beltrí,CarlaMazzeo,NormanNunez-Andrade,EnriqueGabandé-Rodriguez,MariaDoloresLedesma,AlbertoBlázquez,MiguelAngelMartin,JuanManuelFalcón-Pérez
Journal:Cellmetabolism(2015):485--498
TheATP-bindingcassettetransporter-2(ABCA2)regulatesesterificationofplasmamembranecholesterolbymodulationofsphingolipidmetabolism
Authors:WarrenDavis
Journal:BiochimicaetBiophysicaActa(BBA)-MolecularandCellBIOLOGyofLipids(2014):168--179
Ahigh-throughputsphingomyelinaseassayusingnaturalsubstrate
Authors:MiaoXu,KeLiu,NoelSouthall,JuanJMarugan,AlanTRemaley,WeiZheng
Journal:Analyticalandbioanalyticalchemistry(2012):407--414
