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AAT Bioquest/Amplite™ Fluorimetric L-Lactate Assay Kit/13814/200 Tests
| 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.PrepareNADstocksolution(100X):
Add100µLofH2OintothevialofNAD(ComponentC)tomake100XNADstocksolution.
2.PrepareL-lactatestocksolution:
Add200µLofH2Oor1xPBSbufferintothevialofL-lactateStandard(ComponentD)tomake100mML-lacate
standardsolution.
Note:TheunusedD-lacatestandardstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.
3.PrepareserialdilutionsofL-lactatestandard(0to1mM):
3.1 Add10μLofL-lactatestocksolution(fromStep2)into990µLPBSbuffertogenerate1mML-lactatestandardsolution.
Note:DilutedL-lactatestandardsolutionisunstable,andshouldbeusedwithin4hours.
3.2 Take200μLof1mML-lactatestandardsolutiontoperform1:3serialdilutionstoget300,100,30,10,3,1and0μMserialdilutionsofL-lactatestandard.
3.3 AddserialdilutionsofL-lactatestandardandL-lactatecontainingtestsamplesintoasolidblack96-wellmicroplateasdescribedinTables1and2.
Table1LayoutofL-lactatestandardsandtestsamplesinasolidblack96-wellmicroplate
BL | BL | TS | TS | …. | …. |
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Lac1 | Lac1 | …. | …. | …. | …. |
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Lac2 | Lac2 |
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Lac3 | Lac3 |
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Lac4 | Lac4 |
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Lac5 | Lac5 |
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Lac6 | Lac6 |
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Lac7 | Lac7 |
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Note:Lac=L-lactateStandards,BL=BlankControl,TS=TestSamples.
Table2Reagentcompositionforeachwell
L-lactateStandard | BlankControl | TestSample |
SerialDilutions*:50μL | DilutionBuffer:50μL | 50μL |
*Note:AddtheseriallydilutedL-lactatestandardsfrom1μMto1mMintowellsfromLac1toLac7induplicate.
4.PrepareL-lactateassaymixture:
4.1 Add5mLofAssayBuffer(ComponentB)intoonebottleofEnzymeProbe(ComponentA).
4.2 Add50µLNADstocksolution(100X,fromStep1)intothebottleofComponentA (fromStep4.1), andmixwell.
Note:ThisL-lactateassaymixtureisenoughforone96-wellplate.TheunusedL-lactateassaymixtureshouldbedividedintosingleusealiquotsandstoredat-20oC.
5.RunL-lactateassay:
5.1 Add50μLofL-lactate assaymixture(fromStep4.2)toeachwellofL-lactate standard,blankcontrol,andtestsamples(seeStep3.3)tomakethetotalL-lactate assayvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLofL-lactateassaymixtureintoeachwell.
5.2 Incubatethereactionatroomtemperaturefor30minutesto2hours,protectedfromlight.
5.3 MonitorthefluorescenceincreasewithafluorescenceplatereaderatEx/Em=530-570/590-600nm(optimalEx/Em=540/590nm,cutoffat570nm).
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