| 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)into5mLSMaseReactionBuffer(ComponentD),andmixwell.
Note:Thesphingomyelinworkingsolutionshouldbeusedpromptly.
2.Preparesphingomyelinasestandardsand/orsphingomyelinase-containingsamples:
2.1 Add20µLofPBSwith0.1%BSAintothevialofSphingomyelinaseStandard(ComponentF)tomakea10units/mLsphingomyelinasestandardstocksolution.
Note:Theunusedsphingomyelinasestandardstocksolutionshouldbealiquotedandstoredat-20oC.
2.2 Add1µLof10units/mLsphingomyelinasestandardstocksolution(fromStep2.1)into1000µLassaybuffer(ComponentE)togeneratea10mU/mLsphingomyelinasestandard.
Note:Dilutedsphingomyelinasestandardstocksolutionisunstable,shouldbeusedwithin4hours.
2.3 Take500μLof10mU/mLsphingomyelinasestandardtoperform1to2serialdilutionstoget5,2.5,1.25,0.625,0.313,0.156,0.078,and0mU/mLseriallydilutedsphingomyelinasestandards.
2.4 Addtheseriallydilutedsphingomyelinasestandardsand/orsphingomyelinase-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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SMase5 | SMase5 |
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SMase6 | SMase6 |
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SMase7 | SMase7 |
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Note:SMase=SphingomyelinaseStandards,BL=BlankControl,TS=TestSamples.
Table2Reagentcompositionforeachwell
SphingomyelinaseStandards | BlankControl | TestSample |
SerialDilutions:50μL | Assaybuffer:50μL | 50μL |
Note:Addtheseriallydilutedsphingomyelinasestandardsfrom0.078to5mU/mLintowellsfromSMase1toSMase7induplicate.
2.5 Add50μLofsphingomyelinworkingsolution(fromStep1)intoeachwellofsphingomyelinasestandards,blankcontrolandtestsamples(fromStep2.4).
2.6 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.Preparesphingomyelinaseassaymixture:
4.1 Add5mLofAssayBuffer(ComponentE)intothebottleofEnzymeMix(ComponentA),andmixthemwell.
4.2 Add25µLof200XAmplite™Redstocksolution(fromStep3)intothebottleofEnzymeMixsolution(fromStep4.1)tomakethesphingomyelinaseassaymixturebeforestartingtheassay.
Note:Thesphingomyelinaseassaymixtureshouldbeusedpromptlyandkeptfromlight;longerstorageislikelytocausehighassaybackground.
5.Runsphingomyelinaseassay:
5.1 Add50μLofsphingomyelinaseassaymixture(fromStep4.2)intoeachwellofsphingomyelinasestandards,blankcontrol,andtestsamples(fromStep2.4)tomakethetotalsphingomyelinaseassayvolumeof150µL/well.
Note:Fora384-wellplate,add25μLofsample,25μLofsphingomyelinworkingsolution,and25μLofsphingomyelinaseassaymixtureintoeachwell.
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
