| 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.Preparesphingomyelinworkingsolution:
Add50µLofSphingomyelin(ComponentB)to5mLofSMaseReactionBuffer(ComponentD)andmixwell.
Note:Thesphingomyelinworkingsolutionshouldbeusedpromptly.
2.Preparesphingomyelinasestandardsand/orsphingomyelinase-containingsamples:
2.1 Dilutesphingomyelinasesotcksolutionin20mMsodiumacetatebuffer(pH=5.0,notprovidedinthekit).Werecommendtheconcentrationrangefrom10U/mLto0.5U/mL.
Note1:Acidicsphingomyelinasestandard(fromhumanplacenta)isavailablefromSigma-Aldrich(S-5383).
Note2:Dilutedsphingomyelinasestandardsolutionisunstable,andshouldbeusedwithin4hours.
2.2 Addthesphingomyelinasestandardsandsphingomyelinase-containingtestsamplesintoasolidblack96-wellmicroplateasshowninTables1and2.
Note:Treatyourcellsortissuesamplesasdesired.
Table1Layoutofsphingomyelinasestandardsandtestsamplesinasolidblack96-wellmicroplate
BL | BL | TS | TS | …. | …. |
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SMase1 | SMase1 | …. | …. | …. | …. |
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SMase2 | SMase2 |
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SMase3 | SMase3 |
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SMase4 | SMase4 |
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…. | …. |
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…. | …. |
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…. | …. |
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Note:SMase=SphingomyelinaseStandards,BL=BlankControl,TS=TestSamples
Table2Reagentcompositionforeachwell
| SphingomyelinaseStandard | BlankControl | TestSample |
| Serialdilutions:50µL | 20mMsodiumacetatebuffer(pH=5.0):50µL | 50µL |
Note:Addthedilutedsphingomyelinasestandardsinduplicate.
2.3 Add50μLofsphingomyelinworkingsolution(fromStep1)intoeachwellofthesphingomyelinasestandards,blankcontrolandtestsamples(fromStep2.2).
2.4 Incubatethereactionmixtureat37°Cfor2-3hours.
3.Prepare200XAmplite™Redstocksolution:
Add80µLofDMSO(ComponentF)intothevialofAmplite™Red(ComponentC)tomake200X Amplite™stocksolution.
Note1:TheunusedAmplite™Redstocksolutionshouldbealiquotedandstoredat-20oC(protectedfromlight).
Note2:TheAmplite™Redisunstableinthepresenceofthiols(suchasDTT)and2-mercaptoethanol).ThefinalconcentrationofDTTor2-mercaptoethanolinthereactionshouldbelowerthan10μM.Amplite™RedisalsounstableathighpH(>8.5).ThereactionsshouldbeperformedatpH7–8.TheassaybufferatpH7.4isrecommended.
4.Preparesphingomyelinaseassaymixture:
4.1 Add5mLofAssayBuffer(ComponentE)tothebottleofEnzymeMix(ComponentA)andmixwell.
4.2 Add25µLof200XAmplite™Redstocksolution(fromStep3)intothebottleofEnzymeMixsolution(fromStep4.1)tomakethesphingomyelinaseassaymixturebeforestartingtheassay.
Note:Thesphingomyelinaseassaymixtureshouldbeusedpromptlyandkeptfromlight;longerstorageislikelytocausehighassaybackground.
5.Runsphingomyelinaseassay:
5.1 Add50µLofsphingomyelinaseassaymixture(fromStep4.2)intoeachwellofthesphingomyelinasestandards,blankcontrol,andtestsamples(fromStep2.4)tomakethetotalsphingomyelinaseassayvolumeof150µL/well.
Note:Fora384-wellplate,add25μLofsample,25μLofsphingomyelinworkingsolution,and25μLsphingomyelinaseassaymixtureintoeachwell.
5.2 Incubatetheenzymereactionmixturefor1-2hoursatroomtemperature(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
