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AAT Bioquest/Amplite™ Fluorimetric Proteasome 20S Activity Assay Kit *Green Fluorescence*/13456/100 tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 498/520 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | EnzymeDetection PeptidasesandProteases |
| Related | BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1.Preparecells:
1.1 Foradherentcells:Platecellsovernightingrowthmediumat80,000cells/well/90µLfora96-wellplateor20,000cells/well/20µLfora384-wellplate.
1.2 Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandthensUSPendthecellpelletinculturemediumat300,000cells/well/90µLfora96-wellpoly-Dlysineplateor80,000cells/well/20µLfora384-wellpoly-Dlysineplate.Centrifugetheplateat800rpmfor2minuteswithbrakeoffpriortotheexperiments.
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensity.
2.Prepareproteasomeassayloadingsolution:
2.1 Thawallthekitcomponentsatroomtemperaturebeforeuse.
2.2 Make400XProteasomeLLVY-R110Substratestocksolution:Add25µLofDMSO(ComponentC)tothevialofProteasomeLLVY-R110Substrate(ComponentA),andmixwell.
2.3 Makeproteasomeassayloadingsolution:Add25µLof400XProteasomeLLVY-R110Substratestocksolution(fromStep2.2)into10mLofAssayBuffer(ComponentB),andmixwell.
Note:25µLof400XProteasomeLLVY-R110Substratestocksolution(fromStep2.2)and10mLofAssayBuffer(ComponentB)areenoughfor1plate.Aliquotandstoretheunused400XProteasomeLLVY-R110SubstratestocksolutionandAssayBufferat-20oC.Avoidrepeatedfreeze-thawcycles.
3.Runtheproteasomeassay:
3.1 Treatcellswith10µLof10Xtestcompound(fora96-wellplate)or5µLof5Xtestcompound(fora384-wellplate)inPBSordesiredbuffer.Forblankwells(mediumwithoutthecells),addthecorrespondingamountofcompoundbuffer.
3.2 Incubatethecellplatesina5%CO2,37°Cincubatorforadesiredperiodoftime.
Note:Pureproteasomeorcelllysatescanbeuseddirectlyforscreeningtheproteasomeinhibitors.
3.3 Add100µL/well(96-wellplate)or25µL/well(384-wellplate)ofproteasomeassayloadingsolution(fromStep2.3).
3.4 Incubatetheplateat37°Corroomtemperatureforatleast1hour(2hourstoovernight),protectedfromlight.
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalincubationtime.
3.5 Monitorthefluorescenceintensity(topread)atEx/Em=490/525nm.
| References&Citations |  CitationExplorer |
MycobacteriumtuberculosisControlsPhagosomalAcidificationbyTargetingCISH-MediatedSignaling
Authors:ChristopheJQueval,Ok-RyulSong,Jean-PhilippeCarralot,Jean-MichelSaliou,AntoninoBongiovanni,GaspardDeloison,NathalieDeboosère,SamuelJouny,RaffaellaIantomasi,VincentDelorme
Journal:CellReports(2017):3188--3198
Autophagy-mediatedclearanceofubiquitinatedmutanthuntingtinbygrapheneoxide
Authors:PeipeiJin,PengfeiWei,YunjiaoZhang,JunLin,RuiSha,YiHu,JiqianZhang,WeiZhou,HanYao,LiRen
Journal:Nanoscale(2016)
InteractomeanalysisrevealsanovelroleforRAD6intheregulationofproteasomeactivityandlocalizationinresponsetoDNAdamage
Authors:HongliAn,LuYang,ChenWang,ZhixueGan,HaihuiGu,TaoZhang,XinHuang,YanLiu,YufengLi,Shing-JyhChang
Journal:MolecularandCellularBIOLOGy(2016):MCB--00419
L-BMAAinducedERstressandenhancedcaspase12cleavageinhumanneuroblastomaSH-SY5Ycellsatlownonexcitotoxicconcentrations
Authors:OliverOkle,KerstinStemmer,UlrichDeschl,DanielRDietrich
Journal:toxicologicalsciences(2013):217--224
