| 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.PreparePyruvateAssayMixture:
1.1 Thawkitcomponentsatroomtemperaturebeforeuse.
1.2 MakeQuestFluor™Pyruvatesensorstocksolution:Add55μLofDMSO(ComponentE)intoQuestFluor™PyruvateSensor(ComponentA)tomake200XQuestFluor™Pyruvatesensorstocksolution.
1.3 MakeAssayMixture:
1.3.1 Add5mLAssayBuffer(ComponentC)intooneEnzymeMix1bottle(ComponentB1)mixwell.
1.3.2 Add100μLofddH2OintooneEnzymeMix2vial(ComponentB2)mixwell.
1.3.3 Transferentirevial(100μL)ofEnzymeMix2(fromStep1.3.2)and25uLof200Xpyruvatesensorstocksolution(fromStep1.2)intotheEnzymeMix1bottle(fromStep1.3.1)andmixwell.
Note1:Theassaymixtureisnotstable,useitpromptly,andavoiddirectexposure.
Note2:Storeunused200XQuestFluor™Pyruvatesensorstocksolutionat-20oC,avoidlightandrepeatfreeze-thawcycles.
2.Prepareseriallydilutedpyruvatestandardsandtestsamples:
2.1 Preparepyruvatestandard:Add10μLof100mMPyruvate(ComponentD)into990μLofPBS(pH7)toget1mMpyruvatesolution.Thentake100μLof1mMpyruvatestandardsolutioninto900μLPBStomake100µMpyruvatesolution.Andthenperform1:3serialdilutionstoget30,10,3,1,0.3,and0.1μMseriallydilutedpyruvatestandards.
2.2 Addpyruvatecontainingsamplesandseriallydilutedpyruvatestandardsintoasolidblack96-wellmicroplateaccordingtoTables1.
Table1Layoutofpyruvatestandardsandtestsamplesinasolidblack96-wellmicroplate
BL | BL | TS | TS | …. | …. |
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PS1 | PS1 | …. | …. | …. | …. |
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PS2 | PS2 |
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PS3 | PS3 |
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PS4 | PS4 |
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PS5 | PS5 |
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PS6 | PS6 |
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PS7 | PS7 |
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Note1:PS=PyruvateStandard,BL=BlankControl(PBS),TS=TestSample.
Note2:Addtheserialdilutionsofpyruvatestandardfrom0.1μMto100μMintowellsfromPS1toPS7.
3.Runpyruvateassay:
3.1 Add50μLofAssayMixture(fromStep1.3.3)intoeachwellofpyruvatestandard,blankcontrol,andtestsamples(seeStep2.2)tomakethetotalpyruvateassayvolumeof100µL/well.
Note1:Fora384-wellplate,add25μLofsample,25μLofAssaymixture(fromStep1.3)intoeachwell.
Note2:RunthepyruvateassayatpH6.5to7.0.
3.2 Incubatethereactionmixtureatroomtemperaturefor30minutesto1hour.
3.3 MonitorthefluorescenceincreasewithafluorescenceplatereaderatEx/Em=540/590nm(cutoff:570nm).
| References&Citations |  PrinterFriendlyVersion |
1. ShiJ,ZhaoW,ChenY,GuoL,YangL.(2012)Areplaceabledual-enzymecapillarymicroreactorusingmagneticbeadsanditsapplicationforsimultaneousdetectionofacetaldehydeandpyruvate.Electrophoresis,33,2145.
2. MacKenzieJD,YenYF,MayerD,TroppJS,HurdRE,SpielmanDM.(2011)Detectionofinflammatoryarthritisbyusinghyperpolarized13C-pyruvatewithMRimagingandspectroscopy.RADIology,259,414.
3. ZhuA,RomeroR,PettyHR.(2010)AmplexUltraRedenhancesthesensitivityoffluorimetricpyruvatedetection.AnalBiochem,403,123.
4. WolffF,ElKhattABIC,BourdonF,WillemsD.(2009)Comparisonofhigh-performanceliquidchromatographywithfluorescencedetectionversusenzymaticassaytomeasurebloodpyruvateinclinicalpractice.ClinBiochem,42,1099.
5. AlbersMJ,BokR,ChenAP,CunninghamCH,ZierhutML,ZhangVY,KohlerSJ,TroppJ,HurdRE,YenYF,NelsonSJ,VigneronDB,KurhanewiczJ.(2008)Hyperpolarized13Clactate,pyruvate,andalanine:noninvasivebioMarkersforprostatecancerdetectionandgrading.CancerRes,68,8607.
6. ShastriYM,SteinJM.(2008)FaecaltumourpyruvatekinaseM2:notagoodmarkerforthedetectionofcolorectaladenomas.BrJCancer,99,1366;authorreply1367.
7. HaugU,HundtS,BrennerH.(2008)SensitivityandspecificityoffaecaltumourM2pyruvatekinasefordetectionofcolorectaladenomasinalargescreeningstudy.BrJCancer,99,133.
8. LibMY,BrownRM,BrownGK,MarusichMF,CapaldiRA.(2002)DetectionofpyruvatedehydrogenaseE1alpha-subunitdeficienciesinfemalesbyimmunohistochemicaldemonstrationofmosaicisminculturedfibroblasts.JHistochemCytochem,50,877.
9. SchneiderJ,NeuK,GrimmH,VelcovskyHG,WeisseG,EigenbrodtE.(2002)TumorM2-pyruvatekinaseinlungcancerpatients:immunohistochemicaldetectionanddiseasemonitoring.AnticancerRes,22,311.
10. WulkanRW,VerwersR,NeeleM,MantelMJ.(2001)Measurementofpyruvateinbloodbyhigh-performanceliquidchromatographywithfluorescencedetection.AnnClinBiochem,38,554.
