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当前位置: 首页 > 产品中心 > Other_kits > AAT Bioquest/Amplite™ Colorimetric Sphingomyelinase Assay Kit *Blue Color*/13620/200 Tests
商品详细AAT Bioquest/Amplite™ Colorimetric Sphingomyelinase Assay Kit *Blue Color*/13620/200 Tests
AAT Bioquest/Amplite™ Colorimetric Sphingomyelinase Assay Kit *Blue Color*/13620/200 Tests
AAT Bioquest/Amplite™ Colorimetric Sphingomyelinase Assay Kit *Blue Color*/13620/200 Tests
商品编号: 13620
品牌: aatbio
市场价: ¥56560.00
美元价: 33936.00
产地: 美国(厂家直采)
公司:
产品分类: 其它检测试剂盒
公司分类: Other_kits
联系Q Q: 3392242852
电话号码: 4000-520-616
电子邮箱: info@ebiomall.com
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Ex/Em(nm)655/None
MWN/A
CAS#N/A
SolventN/A
StorageF/D/L
CategoryEnzymeDetection
Phosphodiesterases
RelatedBiochemicalAssays
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.
SpectrumAdvancedSpectrumViewer



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-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-containingtestsamplesintowhitewall/clearbottomoraclear96-wellmicroplateasshowninTables1and2.

Note:Treatyourcellsortissuesamplesasdesired.

 

Table1Layoutofsphingomyelinasestandardsandtestsamplesinawhitewall/clearbottom96-wellmicroplate

BL

BL

TS

TS

….

….

 

 

 

 

 

 

SMase1

SMase1

….

….

….

….

 

 

 

 

 

 

SMase2

SMase2

 

 

 

 

 

 

 

 

 

 

SMase3

SMase3

 

 

 

 

 

 

 

 

 

 

SMase4

SMase4

 

 

 

 

 

 

 

 

 

 

SMase5

SMase5

 

 

 

 

 

 

 

 

 

 

SMase6

SMase6

 

 

 

 

 

 

 

 

 

 

SMase7

SMase7

 

 

 

 

 

 

 

 

 

 

       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™Bluestocksolution:

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

Note1:TheunusedAmplite™Bluestocksolutionshouldbealiquotedandstoredat-20oC(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


品牌介绍
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会不断研发新产品,快速地丰富各个领域的产品。 1)提供反应探针和发光探针,生物素和端粒酶能够应用于标记药物小分子和生物聚合物,如蛋白、核酸以及其他碳水化合物; 2)研究并生产荧光和发光探针用于检测蛋白,核酸和活细胞; 3)不断推出新型的荧光和发光探针用于检测多种酶,特别是检测水解酶和氧化还原酶类; 4)致力于开发用于信号转导研究的试剂; 5)提供生理和神经探针,特别是钙离子指示剂和膜电位探针。
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淋巴细胞信号传导 GPCR抗体 羰基活性生物素 细胞代谢 胺反应生物素 链霉亲和素结合物 生物素化抗IgGs 乙酰基特异性抗体 细胞骨架抗体
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