| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 571/585 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellBIOLOGy CellMetabolism |
| Related | RedoxEnzymes BiochemicalAssays |
1.Prepareseriallydilutedoxaloacetatestandardsandtestsamples:
1.1 Add100µLofddH2Ointooxaloacetatestandard(ComponentE)tomake100mMoxaloacetatestandardsolution.
1.2 Prepareoxaloacetatestandarddilutions:Add10μLof100mMoxaloacetate(fromStep1.1)into990μLofassaybuffer(ComponentC)toget1mMoxaloacetatesolution.Take100μLof1mMoxaloacetatestandardsolutioninto900μLassaybuffertomake100µMoxaloacetatesolution.Perform1:3serialdilutionstogetapproximately30,10,3,1,0.3,and0.1μMseriallydilutedoxaloacetatestandards.
1.3 Add50μLofoxaloacetatecontainingsamplesandseriallydilutedoxaloacetatestandardsintoasolidblack96-wellmicroplateaccordingtoTables1.
Table1Layoutofoxaloacetatestandardsandtestsamplesinasolidblack96-wellmicroplate
BL | BL | TS | TS | …. | …. |
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OAA1 | OAA1 | …. | …. | …. | …. |
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OAA2 | OAA2 |
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OAA3 | OAA3 |
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OAA4 | OAA4 |
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OAA5 | OAA5 |
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OAA6 | OAA6 |
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OAA7 | OAA7 |
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Note1:OAA=OxaloacetateStandard,BL=BlankControl(assaybuffer),TS=TestSample.
Note2:Addtheserialdilutionsofoxaloacetatestandardfrom0.1μMto100μMintowellsfromOAA1toOAA7.
2.PrepareoxaloacetateAssayMixture:
2.1 MakeAmplite™Redsubstratestocksolution(200X):Add50μLofDMSO(ComponentF)intoAmplite™Redsubstrate(ComponentA)tomake200XAmplite™Redsubstratestocksolution.
2.2 Makeoxaloacetatedecarboxcylase(OAC)stocksolution(100X):Add50μLofddH2Ointooxaloacetatedecarboxcylase(ComponentD)tomake100Xoxaloacetatedecarboxcylasestocksolution.
2.3 MakeAssayMixture:
2.3.1Add5mLAssayBuffer(ComponentC)intooneEnzymeMix1bottle(ComponentB1)andmixwell.
2.3.2Add100μLofddH2OintooneEnzymeMix2vial(ComponentB2)andmixwell.
2.3.3Transfer25μLof200XAmplite™Redstocksolution(fromStep2.1),50μLOACstocksolution(fromstep2.2),andentirevial(100μL)ofEnzymeMix2(fromStep2.3.2)intotheEnzymeMix1bottle(fromStep2.3.1)andmixwell.
Note1:Theassaymixtureisnotstable,useitpromptly,andavoiddirectexposuretolight.
Note2:Storeunused200XAmplite™Redstocksolutionat-20oC,avoidlightandrepeatedfreeze-thawcycles.
3.Runoxaloacetateassay:
3.1 Add50μLofAssayMixture(fromStep2.3)intoeachwellofoxaloacetatestandard,blankcontrol,andtestsamples(seeStep1.3)tomakethetotaloxaloacetateassayvolumeof100µL/well.
Note1:Fora384-wellplate,add25μLofsample,25μLofAssaymixtureintoeachwell.
Note2:RuntheoxaloacetateassayatpH6.5to7.0.
3.2 Incubatethereactionmixtureatroomtemperaturefor30minutesto1hour.
3.3 MonitorthefluorescenceincreasewithafluorescenceplatereaderatEx/Em=540/590nm(cutoff:570nm).
| References&Citations |  PrinterFriendlyVersion |
1. KatoS,IkutaT,HemmiH,TakahashiS,KeraY,YoshimuraT.(2012)EnzymaticassayforD-asparticacidusingD-aspartateoxidaseandoxaloacetatedecarboxylase.BiosciBiotechnolBiochem,76,2150.
2. PentyalaSN,BenjaminWB.(1995)EffectofoxaloacetateandphosphorylationonATP-citratelyaseactivity.Biochemistry,34,10961.
3. LaplanteA,ComteB,DesRosiersC.(1995)Assayofbloodandtissueoxaloacetateandalpha-ketoglutaratebyisotopedilutiongaschromatography-massspectrometry.AnalBiochem,224,580.
4. LeikinYN,ZharovaTV,TjulinaOV.(1993)NoveloxaloacetateeffectonmitochondrialCa2+movement.FEBSLett,331,35.
5. Arias-MendozaF,PinaE.(1991)Asensitivemultienzymaticassayforthemeasurementofpyruvate,dihydroxyacetonephosphate,oxaloacetate,andacetoacetateinclearextractsfrombiologicalsamples.PrepBiochem,21,211.
6. KurkijarviK,VierijokiT,KorpelaT.(1990)Flow-injectionanalysisofaminoacidsandtheirmetabolitesbyimmobilizedvitaminB6-dependentenzymes.SensitivedeterminationofL-aspartate,L-glutamate,2-oxoglutarate,andoxaloacetate.AnnNYAcadSci,585,394.
7. HaasRH,ThompsonG,MorrisB,ConrightK,AndrewsT.(1988)Pyruvatedehydrogenaseactivityinosmoticallyshockedratbrainmitochondria:stimulationbyoxaloacetate.JNeuRochem,50,673.
8. WalkerGH,KuMS,EdwardsGE.(1986)Activityofmaizeleafphosphoenolpyruvatecarboxylaseinrelationtotautomerizationandnonenzymaticdecarboxylationofoxaloacetate.ArchBiochemBiophys,248,489.
9. NimmoHG.(1986)KineticmechanismofEscherichiacoliisocitratedehydrogenaseanditsinhibitionbyglyoxylateandoxaloacetate.BiochemJ,234,317.
10. SimpsonJ.(1981)TheinteractionofdithiothreitolandacetylcoenzymeAinarADIochemicalassayforratbrainATP:citrateoxaloacetatelyase.JNeurochem,37,100.
