>
产品中心 >
Other_kits >
AAT Bioquest/Amplite™ Fluorimetric Glutamate Oxidase Assay Kit *Red Fluorescence*/11302/200 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 571/585 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | SmallMoleculeDetection DiagnosticMolecules |
| Related | RedoxEnzymes BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1.Preparestocksolutions:
1.1 Amplite™Redstocksolution(250X):Add40µLofDMSO(ComponentF)intothevialofAmplite™Red(ComponentA).Thestocksolutionshouldbeusedpromptly.Anyremainingsolutionshouldbealiquotedandfrozenat-20°C.
Note1:Avoidrepeatedfreeze-thawcycles.
Note2:TheAmplite™Redisunstableinthepresenceofthiolssuchasdithiothreitol(DTT)and2-mercaptoethanol.ThefinalconcentrationofDTTor2-mercaptoethanolinthereactionshouldbenohigherthan10μM.TheAmplite™RedisalsounstableathighpH(>8.5).Therefore,thereactionshouldbeperformedatpH7–8.Theprovidedassaybuffer,pH7.4,isrecommended.
1.2 HRPstocksolution(400X):Add200µLofAssayBuffer(ComponentB)intothevialofHorserADIshPeroxidase(ComponentC).
Note:TheunusedHRPstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20°C.
1.3 GlutamicAcidstocksolution(400X):Add1.0mLofddH2OintothevialofGlutamicAcid(ComponentD)tomake400Xglutamicacidstocksolution.
Note:Theunusedglutamicacidstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20°C.
1.4 150mU/mLGlutamateOxidase(GO)solution:Add100µLofAssayBuffer(ComponentB)intothevialofGlutamateOxidaseStandard(lyophilized)(ComponentE)tomake150mU/mLGlutamateOxidase(GO)solution.
Note:Theunusedglutamateoxidasesolutionshouldbedividedintosingleusealiquotsandstoredat-20°C.
2.Prepareassaymixture:
Prepareassaymixtureaccordingtothefollowingtablesandprotectfromlight.
Table1.Assaymixtureforone96-wellplate(2X)
Components | Volume |
Amplite™Redstocksolution(250X,fromStep1.1) | 20µL |
HRP(400X,fromStep1.2) | 12.5µL |
GlutamicAcid(400X,fromStep1.3) | 12.5µL |
AssayBuffer(ComponentB) | 5mL |
Totalvolume | 5.07mL |
3.PrepareseriallydilutedGOstandards(0to10mU/mL):
3.1 Add30μLof150mU/mLGOstocksolution(fromStep1.4)into420μLofAssayBuffer(ComponentB)toget10mU/mLGOstandardsolution.
3.2 Take150μLof10mU/mLGOstandardsolutiontoperform1:3serialdilutionstoget3,1,0.3,0.1,0.03,0.0,1and0mU/mLseriallydilutedGOstandards.
3.3 AddGOstandardsand/orGO-containingtestsamplesintoablackwall/solidbottom96-wellmicroplateasdescribedinTables2and3
Table2.LayoutofGOstandardsandtestsamplesinasolidblack96-wellmicroplate
BL | BL | TS | TS | …. | …. |
|
|
|
|
|
|
GO1 | GO1 | …. | …. | …. | …. |
|
|
|
|
|
|
GO2 | GO2 |
|
|
|
|
|
|
|
|
|
|
GO3 | GO3 |
|
|
|
|
|
|
|
|
|
|
GO4 | GO4 |
|
|
|
|
|
|
|
|
|
|
GO5 | GO5 |
|
|
|
|
|
|
|
|
|
|
GO6 | GO6 |
|
|
|
|
|
|
|
|
|
|
GO7 | GO7 |
|
|
|
|
|
|
|
|
|
|
Note:GO=glutamateoxidasestandards,BL=blankcontrol,TS=testsamples.
Table3.Reagentcompositionforeachwell
GOStandard | BlankControl | TestSample |
SerialDilutions*(50μL) | AssayBuffer(ComponentB):50μL | 50μL |
*Note1:Addtheseriallydilutedglutamateoxidasestandardsfrom0.01mU/mLto10mU/mLintoeachwellfromGO1toGO7induplicate.
Note2:HighconcentrationofGOmaycausereducedfluorescencesignalduetotheoveroxidationofAmplite™Red(toanon-fluorescentproduct).
4.RunGOassay:
4.1 Add50μLofassaymixture(fromStep2)intoeachwelloftheGOstandard,blankcontrol,andtestsamples(seeStep3,Table2)tomakethetotalGOassayvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLofassaymixtureintoeachwell.
4.2 Incubatethereactionfor30to60minutesatroomtemperature,protectedfromlight.
4.3 MonitorthefluorescenceintensitywithafluorescenceplatereaderatEx/Em=530-570nm/590-600nm(optimalEx/Em=540/590nm),cutoff=570nm.
Note:Thecontentsoftheplatecanalsobetransferredtoawhiteclearbottomplateandreadbyanabsorbancemicroplatereaderatthewavelengthof576±5nm.Theabsorptiondetectionhaslowersensitivitycomparedtofluorescencereading.
| References&Citations |  PrinterFriendlyVersion |
1. ArimaJ,SasakiC,SakaguchiC,MizunoH,TamuraT,KashimaA,KusakabeH,SugioS,InagakiK.(2009)StructuralcharacterizationofL-glutamateoxidasefromStreptomycessp.X-119-6.FebsJ,276,3894.
2. DharSS,LiangHL,Wong-RileyMT.(2009)Nuclearrespiratoryfactor1co-regulatesAMPAglutamatereceptorsubunit2andcytochromecoxidase:tightcouplingofglutamatergictransmissionandenergymetabolisminneurons.JNeuRochem,108,1595.
3. DharSS,Wong-RileyMT.(2009)Couplingofenergymetabolismandsynaptictransmissionatthetranscriptionallevel:roleofnuclearrespiratoryfactor1inregulatingbothcytochromecoxidaseandNMDAglutamatereceptorsubunitgenes.JNeurosci,29,483.
4. LoaneDJ,StoicaBA,Pajoohesh-GanjiA,ByrnesKR,FadenAI.(2009)Activationofmetabotropicglutamatereceptor5modulatesmicroglialreactivityandneurotoxicitybyinhibitingNADPHoxidase.JBiolChem,284,15629.
5. OkumuraW,MorideraN,KanazawaE,ShojiA,Hirano-IwataA,SugawaraM.(2009)VisualizingL-glutamatefluxesinacutehippocampalsliceswithglutamateoxidase-immobilizedcoverslips.AnalBiochem,385,326.
6. DurrKL,KoepkeJ,HellwigP,MullerH,AngererH,PengG,OlkhovaE,RichterOM,LudwigB,MichelH.(2008)AD-pathwaymutationdecouplestheParacoccusdenitrificanscytochromecoxidasebyalteringtheside-chainorientationofadistantconservedglutamate.JMolBiol,384,865.
7. YangK,ZhangJ,VakkasogluAS,HielscherR,OsborneJP,HempJ,MiyoshiH,HellwigP,GennisRB.(2007)Glutamate107insubunitIofthecytochromeBDquinoloxidasefromEscherichiacoliisprotonatedandnearthehemed/hemeb595binuclearcenter.Biochemistry,46,3270.
8. BasuAK,ChattopadhyayP,RoychudhuriU,ChakrabortyR.(2006)DevelopmentofbiosensorbasedonimmobilizedL-glutamateoxidasefordeterminationofmonosodiumglutamateinfood.IndianJExpBiol,44,392.
9. BasuAK,ChattopadhyayP,RoychudhuriU,ChakrabortyR.(2006)Abiosensorbasedonco-immobilizedL-glutamateoxidaseandL-glutamatedehydrogenaseforanalysisofmonosodiumglutamateinfood.BiosensBioelectron,21,1968.
10. McMahonCP,RocchittaG,SerraPA,KirwanSM,LowryJP,O"NeillRD.(2006)Theefficiencyofimmobilisedglutamateoxidasedecreaseswithsurfaceenzymeloading:anelectrostaticeffect,andreversalbyapolycationsignificantlyenhancesbiosensorsensitivity.Analyst,131,68.
