Overview

PrinterFriendlyVersion

Ex/Em(nm)	655/None
MW	N/A
CAS#	N/A
Solvent	N/A
Storage	F/D/L
Category	EnzymeDetection Phosphodiesterases
Related	BiochemicalAssays

Sphingomyelinase(SMase)isanenzymethatisresponsIBLeforcleavingsphingomyelin(SM)tophosphocholineandceramide.ActivationofSMasesincellsplaysanimportantroleinthecellularresponses.Fivetypesofsphingomyelinase(SMase)havebeenidentifiedbasedontheircationdependenceandpHoptimaofaction.TheyarelysosomalacidSMase,secretedzinc-dependentacidSMase,magnesium-dependentneutralSMase,magnesium-independentneutralSMase,andalkalineSMase.Amongthefivetypes,thelysosomalacidicSMaseandthemagnesium-dependentneutralSMaseareconsideredmajorcandidatesfortheproductionofceramideinthecellularresponsetostress.OurAmplite™ColorimetricSphingomyelinaseAssayKitprovidesasensitivemethodfordetectingneutralSMaseactivityorscreeningitsinhibitors.ThekitusesAmplite™Blueasacolorimetricprobetoindirectlyquantifythephosphocholineproducedfromthehydrolysisofsphingomyelin(SM)bysphingomyelinase(SMase).ItcanbeusedformeasuringtheSMaseactivityinblood,cellextractsorothersolutions.Theabsorbanceoflightat655nmisproportionaltotheformationofphosphocholine,thereforetotheSMaseactivity.Thekitisanoptimized"mixandread"assaythatiscompatiblewithHTSliquidhandlinginstruments.

Spectrum

AdvancedSpectrumViewer

Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.Preparesphingomyelinworkingsolution:

Add50µLofSphingomyelin(ComponentB)into5mLSMaseReactionBuffer(ComponentD),andmixwell.

Note:Thesphingomyelinworkingsolutionshouldbeusedpromptly.

2.Preparesphingomyelinasestandardsand/orsphingomyelinase-containingsamples:

 

2.1   Add20µLofPBSwith0.1%BSAintothevialofSphingomyelinaseStandard(ComponentF)tomakea10units/mLsphingomyelinasestandardstocksolution.

Note:Theunusedsphingomyelinasestandardstocksolutionshouldbealiquotedandstoredat-20^oC.

 

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-containingtestsamplesintowhitewall/clearbottomoraclear96-wellmicroplateasshowninTables1and2.

Note:Treatyourcellsortissuesamplesasdesired.

 

Table1Layoutofsphingomyelinasestandardsandtestsamplesinawhitewall/clearbottom96-wellmicroplate

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

 

Table2Reagentcompositionforeachwell

Note:Addtheseriallydilutedsphingomyelinasestandardsfrom0.078to5mU/mLintowellsfromSMase1toSMase7induplicate.

 

2.5   Add50μLofsphingomyelinworkingsolution(fromStep1)intoeachwellofsphingomyelinasestandards,blankcontrolandtestsamples(fromStep2.4).

2.6   Incubatethereactionmixtureat37°Cfor1-2hours.

3.Prepare200XAmplite™Bluestocksolution:

Add100µLofDMSO(ComponentG)intothevialofAmplite™Blue(ComponentC)tomake200XAmplite™Bluestocksolution.

Note1:TheunusedAmplite™Bluestocksolutionshouldbealiquotedandstoredat-20^oC(keptfromlight).

Note2:TheAmplite™Blueisunstableinthepresenceofthiols(suchasDTTand2-mercaptoethanol).ThefinalconcentrationofDTTor2-mercaptoethanolinthereactionshouldbelowerthan10μM.Amplite™BlueisalsounstableathighpH(>8.5).ThereactionsshouldbeperformedatpH7-8.pH7.4isrecommendedfortheassaybuffer.

4.Preparesphingomyelinaseassaymixture:

4.1   Add5mLofAssayBuffer(ComponentE)intothebottleofEnzymeMix(ComponentA),andmixthemwell.

4.2   Add50µLof200XAmplite™Bluestocksolution(fromStep3)intothebottleofEnzymeMixsolution(fromStep4.1tomakethesphingomyelinaseassaymixturebeforestartingtheassay.

Note1:Thesphingomyelinaseassaymixtureshouldbeusedpromptlyandkeptfromlight;longerstorageislikelytocausehighassaybackground.

Note2:Thecloudinessofthemixtureisnormal;itwillnotinterferewiththeassayperformance.

 

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   Monitortheabsorbanceincreasewithanabsorbancemicroplatereaderat655nm.

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

美国AAT Bioquest Inc.(前身是ABD Bioquest，Inc.)是一家为从事生命科学研究、诊断研发及药物开发的科学家研发、生产和销售生物分析研究试剂和试剂盒的公司。公司致力于光谱学检测领域，包括吸收（颜色），荧光和发光技术。AAT Bioquest的产品帮助全世界的科学家和生物医药研究者更好的了解生物化学，免疫学，细胞生物学和分子生物学等领域。AAT Bioquest会不断介绍新产品，快速的丰富各个领域的产品。作为AAT Bioquest Inc.的中国区域代理，蚂蚁淘科技为中国客户提供光谱学检测领域，包括底物显色、荧光和发光技术等全系列解决方案。       １、更具品质保障的AAT Bioquest产品（AAT Bioquest作为全球专业的光谱学检测产品生产商，能提供最具性价比的产品）； ２、更高价格优势和性价比的选择，我们将一如既往的为客户和代理商提供最具价格竞争力的产品和全面的售后保障（作为全国总代理，我们能提供最大的价格优惠和更全面的技术支持） ３、更快捷的到货时间，我们将以更短的订货周期，更快捷的国际物流以及报关时间为您提供最节省时间的供货保证（蚂蚁淘具有独立的进出口和报关权，因此提供更快捷的到货保障）；