| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 575/None |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellBIOLOGy CellMetabolism |
| Related | RedoxEnzymes BiochemicalAssays |
1.PrepareL-AlanineAssayMixture:
1.1 Thawkitcomponentsatroomtemperaturebeforeuse.
1.2 MakeQuestFluor™L-AlanineSensorStockSolution:Add55μLofDMSO(ComponentE)intoQuestFluor™L-AlanineSensor(ComponentA)tomake200XQuestFluor™L-alaninesensorstocksolution.
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),and25uLof200XL-alanine sensorstocksolution(fromStep1.2)intotheEnzymeMix1bottle(fromStep1.3.1)andmixwell.
Note1:Theassaymixtureisnotstable,useitpromptly,andavoiddirectexposuretolight.
Note2:Storeunused200XQuestFluor™L-alaninesensorstocksolution(fromStep1.2)at-20oC,avoidlightandrepeatedfreeze-thawcycles.
2.PrepareSeriallyDilutedL-AlanineStandardsandTestSamples:
2.1 PrepareL-alaninestandard:Add10μLof100mML-alanine(ComponentD)into990μLof PBS(pH7.0)toget1mML-alaninesolution.Add100μLof1mML-alaninestandardsolutioninto900μLPBStomake100µML-alaninesolution.Perform1:2serialdilutionstoget50,25,12.5,6.25,3.125and1.563μMseriallydilutedL-alaninestandards.
2.2 AddL-alaninecontainingsamplesandseriallydilutedL-alaninestandardsintoawhiteclear96-wellmicroplateaccordingtoTables1.
Table1LayoutofL-alaninestandardsandtestsamplesinawhiteclear96-wellmicroplate.
BL | BL | TS | TS | …. | …. |
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AS1 | AS1 | …. | …. | …. | …. |
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AS2 | AS2 |
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AS3 | AS3 |
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AS4 | AS4 |
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AS5 | AS5 |
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AS6 | AS6 |
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AS7 | AS7 |
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Note1:AS=L-AlanineStandard,BL=BlankControl(PBS),TS=TestSample.
Note2:AddtheserialdilutionsofL-Alaninestandardfrom1.5μMto100μMintowellsfromAS1toAS7.
3.RunL-alanineassay:
3.1 Add50μLofAssayMixture(fromStep1.3.3)intoeachwellofL-alaninestandard,blankcontrolandtestsamples(seeStep2.2)tomakethetotalL-alanineassayvolumeof100µL/well.
Note1:Fora384-wellplate,add25μLofsample,25μLofAssaymixture(fromStep1.3)intoeachwell.
Note2:RuntheL-alanineassayatpH6.5to7.0.
3.2 Incubatethereactionmixtureat37oCfor30minutesto1hour.
3.3 Monitortheabsorbanceincreasewithanabsorbanceplatereaderat575nm.
| References&Citations |  PrinterFriendlyVersion |
1. GloverWB,LibertoCM,McNeilWS,BanackSA,ShipleyPR,MurchSJ.(2012)Reactivityofbeta-methylamino-L-alanineincomplexsamplematrixescomplicatingdetectionandquantificationbymassspectrometry.AnalChem,84,7946.
2. GuidelliEJ,RamosAP,ZaniquelliME,NicolucciP,BaffaO.(2012)Synthesisandcharacterizationofsilver/alaninenanocompositesforrADIationdetectioninmedicalapplications:theinfluenceofparticlesizeonthedetectionproperties.Nanoscale,4,2884.
3. PerezMA,SousaSF,OliveiraEF,FernandesPA,RamosMJ.(2011)Detectionoffarnesyltransferaseinterfacehotspotsthroughcomputationalalaninescanningmutagenesis.JPhysChemB,115,15339.
4. TangZ,LiuP,MaC,YangX,WangK,TanW,LvX.(2011)Molecularbeaconbasedbioassayforhighlysensitiveandselectivedetectionofnicotinamideadeninedinucleotideandtheactivityofalanineaminotransferase.AnalChem,83,2505.
5. HuangFR,ZhangJ,LuoYH,LiSP,ZhengSF,ChenXD.(2010)[Rapiddetectionofalanineaminotransferasewithnear-infraredspectroscopy].GuangPuXueYuGuangPuFenXi,30,2620.
6. WysockaM,LesnerA,GuzowK,KulczyckaJ,LegowskaA,WiczkW,RolkaK.(2010)Highlyspecificsubstratesofproteinase3containing3-(2-benzoxazol-5-yl)-l-alanineandtheirapplicationfordetectionofthisenzymeinhumanserum.AnalChem,82,3883.
7. SchwimmerJB,DunnW,NormanGJ,PardeePE,MiddletonMS,KerkarN,SirlinCB.(2010)SAFETYstudy:alanineaminotransferasecutoffvaluesaresettoohighforreliabledetectionofpediatricchronicliverdisease.Gastroenterology,138,1357.
8. SuYS,LinYP,ChengFC,JenJF.(2010)In-capillaryderivatizationandstackingelectrophoreticanalysisofgamma-aminobutyricacidandalanineinteasamplestoredeemthedetectionafterdilutiontodecreasematrixinterference.JAgricFoodChem,58,120.
9. MiyoshiY,HamaseK,TojoY,MitaM,KonnoR,ZaitsuK.(2009)DeterminationofD-serineandD-alanineinthetissuesandphysiologicalfluidsofmicewithvariousD-amino-acidoxidaseactivitiesusingtwo-dimensionalhigh-performanceliquidchromatographywithfluorescencedetection.JChromatogrBAnalytTechnolBiomedLifeSci,877,2506.
10. GlinghammarB,RafterI,LindstromAK,HedbergJJ,AnderssonHB,LindblomP,BergAL,CotgreaveI.(2009)Detectionofthemitochondrialandcatalyticallyactivealanineaminotransferaseinhumantissuesandplasma.IntJMolMed,23,621.
