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Ex/Em(nm)	571/585
MW	N/A
CAS#	N/A
Solvent	N/A
Storage	F/D/L
Category	EnzymeDetection Phosphodiesterases
Related	BiochemicalAssays

Sphingomyelinase(SMase)isanenzymethatisresponsIBLeforcleavingsphingomyelin(SM)tophosphocholineandceramide.ActivationofSMasesplaysanimportantroleinthecellularresponsesuchasregulationofcellgrowth,celldifferentiation,cellcyclearrestandprogrammedcelldeath.Fivetypesofsphingomyelinase(SMase)havebeenidentifiedbasedontheircationdependenceandpHoptimaofaction,includinglysosomalacidSMase,secretedzinc-dependentacidSMase,magnesium-dependentneutralSMase,magnesium-independentneutralSMaseandalkalineSMase.Amongthem,thelysosomalacidicSMaseandthemagnesium-dependentneutralSMaseareconsideredtobethemajorfactorsfortheproductionofceramideincellularstressresponses.OurAmplite™FluorimetricAcidicSphingomyelinaseAssayKitprovidesoneofthemostsensitivemethodsfordetectingacidicSMaseactivityorscreeningitsinhibitors.ThekitusesAmplite™Redasafluorogenicprobetoindirectlyquantifythephosphocholineproducedfromthehydrolysisofsphingomyelin(SM)bysphingomyelinase(SMase).ItcanbeusedformeasuringtheSMaseactivityinblood,cellextractsorothersolutions.ThefluorescenceintensityofAmplite™Redisproportionaltotheformationofphosphocholine,thereforetotheSMaseactivity.Thekitisanoptimized"mixandread"assaycompatiblewithHTSliquidhandlinginstruments.

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Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

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

       Note:SMase=SphingomyelinaseStandards,BL=BlankControl,TS=TestSamples

 

Table2Reagentcompositionforeachwell

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-20^oC(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

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