| 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 |
1.1Add100µLofddH2OintoAspartateStandardvial(ComponentE)tomake10mMaspartatestocksolution.
1.2Prepareaspartatestandarddilutions:Add10µLof10mMaspartatestandard(fromstep1.1)into990µLof1xPBSbuffertoget100µMaspartatesolution.Thenperform1:3serialdilutionsin1xPBSbuffertogetapproximately30,10,3,1,0.3,and0.1µMseriallydilutedaspartatestandards.
1.3Addaspartatecontainingsamplesandseriallydilutedaspartatestandardsintoasolidblack96-wellmicroplateaccordingtoTable1.
| BL | BL | TS | TS | ... | ... | ||||||
| ASP1 | ASP1 | ... | ... | ... | ... | ||||||
| ASP2 | ASP2 | ||||||||||
| ASP3 | ASP3 | ||||||||||
| ASP4 | ASP4 | ||||||||||
| ASP5 | ASP5 | ||||||||||
| ASP6 | ASP6 | ||||||||||
| ASP7 | ASP7 |
Note1:ASP=AspartateStandard,BL=BlankControl(1xPBSbuffer),TS=TestSample.
Note2:Addtheserialdilutionsofaspartatestandardsfrom0.1µMto100µMintowellsfromASP1toASP7.
2.PrepareAspartateAssayMixture:
2.1MakeAmplite™Redsubstratestocksolution:Add50µLofDMSO(ComponentF)intoAmplite™Redsubstrate(ComponentA)tomake200XAmplite™Redsubstratestocksolution.
2.2MakeConversionMixstocksolution:Add50µLofddH2OintoConversionMix(ComponentD)tomake100XConversionMixstocksolution.
2.3MakeAssayMixture:
2.3.1Add5mLAssayBuffer(ComponentC)intooneEnzymeMix1bottle(ComponentB1)mixwell.
2.3.2Add100µLofddH2OintooneEnzymeMix2vial(ComponentB2)mixwell.
2.3.3Transferentirevial(100µL)ofEnzymeMix2(fromStep2.3.2)and25uLof200XAmplite™Redsubstratestocksolution(fromStep2.1),50µLof100XConversionMixstocksolution(fromStep2.2)intotheEnzymeMix1bottle(fromStep2.3.1)andmixwell.
Note2:Storeunused200XAmplite™Redstocksolutionat-20°C,avoidlightandrepeatedfreeze-thawcycles.
3.Runaspartateassay:
3.1Add50µLofAssayMixture(fromStep2.3)intoeachwellofaspartatestandard,blankcontrol,andtestsamples(seeStep1.3)tomakethetotalaspartateassayvolumeof100µL/well.
Note1:Fora384-wellplate,add25µLofsample,25µLofAssaymixtureintoeachwell.
Note2:RuntheaspartateassayatpH6.5to7.0.
3.2Incubatethereactionmixtureat37°Cfor20-30minutes.
3.3MonitorthefluorescenceincreasewithafluorescenceplatereaderatEx/Em=540/590nm(cutoff:570nm).
| References&Citations |  PrinterFriendlyVersion |
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7. SchmidtJ.(2003)Feasibilitystudy:fastliquidchromatography-massspectrometryforthequantificationofasparticacidinanaspartatedrug.AnalBioanalChem,377,1120.
8. AylinF,KonuklarS,AviyenteV.(2003)Modellingthehydrolysisofsuccinimide:formationofaspartateandreversibleisomerizationofasparticacidviasuccinimide.OrgBiomolChem,1,2290.
9. CuestaR,GlidewellC,LopezR,LowJN.(2003)PotassiumN-(6-amino-3,4-dihydro-3-methyl-5-nitroso-4-oxopyrimidin-2-yl)-(S)-aspartateN-(6-amino-3,4-dihydro-3-methyl-5-nitroso-4-oxopyrimidin-2-yl)-(S)-asparticacid4.88-hydrate:atwo-dimensionalcoordinationpolymer.ActaCrystallogrC,59,m315.
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