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AAT Bioquest/Amplite™ Fluorimetric Glutamic Acid Assay Kit *Red Fluorescence*/10054/200 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 |
1.PrepareNADPstocksolution(200X):
Add100µLofDilutionBuffer(ComponentE)intothevialofNADP(ComponentC)tomake200XNADPstocksolution.
Note:TheunusedNADPstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.
2.Prepareglutamicacidstocksolution:
Add200µLofDilutionBuffer(ComponentE)intothevialofGlutamicAcid(ComponentD)tomake100mMglutamicacidstocksolution.
Note:Theunusedglutamicacidstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.
3.Prepareglutamicacidassaymixture:
3.1 Add10mLofAssayBuffer(ComponentB)intothebottleofEnzymeMixture(ComponentA).
3.2 Add50µL200XNADPstocksolution(fromStep1)intotheEnzymeMixturebottle(fromStep3.1),andmixthemwell.
Note:Thisglutamicacidassaymixtureisenoughfortwo96-wellplates.Theunusedglutamicacidassaymixtureshouldbedividedintosingleusealiquotsandstoredat-20oC.
4.Prepareseriallydilutedglutamicacidstandards(0to1mM):
4.1Add10μLofglutamicacidstocksolution(fromStep2)into990µLDilutionBuffer(ComponentE)togenerate1mMglutamicacidstandardsolution.Note:Dilutedglutamicacidstandardsolutionisunstable.Usewithin4hours.
4.2 Take200μLof1mMglutamicacidstandardsolutiontoperform1:3serialdilutionstoget300,100,30,10,3,1and0μMseriallydilutedglutamicacidstandards.
4.3 Addseriallydilutedglutamicacidstandardsandglutamicacidcontainingtestsamplesintoasolidblack96-wellmicroplateasdescribedinTables1and2.
Table1.Layoutofglutamicacidstandardsandtestsamplesinasolidblack96-wellmicroplate
BL | BL | TS | TS | …. | …. |
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GLU1 | GLU1 | …. | …. | …. | …. |
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GLU2 | GLU2 |
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GLU3 | GLU3 |
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GLU4 | GLU4 |
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GLU5 | GLU5 |
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GLU6 | GLU6 |
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GLU7 | GLU7 |
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Note:GLU=GlutamicAcidStandards,BL=BlankControl,TS=TestSamples.
Table2.Reagentcompositionforeachwell
GlutamicAcidStandards | BlankControl | TestSample |
SerialDilutions*:50μL | DilutionBuffer:50μL | 50μL |
*Note:Addtheseriallydilutedglutamicacidstandardsfrom1μMto1mMintowellsfromGLU1toGLU7induplicate.
5.Runglutamicacidassay:
5.1 Add50μLofglutamicacidassaymixture(fromStep3)intoeachwellofglutamicacidstandard,blankcontrol,andtestsamples(seeStep4.3)tomakethetotalglutamicacidassayvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLofglutamicacidassaymixtureintoeachwell.
5.2 Incubatethereactionatroomtemperaturefor30minutesto2hours,protectedfromlight.
5.3 MonitorthefluorescenceincreasebyusingafluorescenceplatereaderatEx/Em=530-570/590-600nm(optimalEx/Em=540/590nm),cutoff=570nm.
Note:Thecontentsoftheplatecanalsobetransferredtoawhiteclearbottomplateandreadbyabsorbancemicroplatereaderatthewavelengthof576±5nm.Theabsorptiondetectionhaslowersensitivitycomparedtothefluorescencereading.
| References&Citations |  CitationExplorer |
Presenilin-1/γ-secretasecontrolsglutamaterelease,tyrosinephosphorylation,andsurfaceexpressionofN-methyl-d-aspartatereceptor(NMDAR)subunitGluN2B
Authors:ZhaoXuan,GaelBarthet,JunichiShioi,JindongXu,AnastasiosGeorgakopoulos,JulienBruban,NikolaosKRobakis
Journal:JournalofBiologicalChemistry(2013):30495--30501
