| 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.PrepareNADstocksolution(100X):
Add100µLofH2OintothevialofNAD(ComponentC)tomake100XNADstocksolution.
2.PrepareD-lactatestocksolution:
Add200µLofH2Oor1xPBSbufferintothevialofD-lactateStandard(ComponentD)tomake100mMD-lacatestandardsolution.
Note:TheunusedD-lacatestandardstocksolutionshouldbedividedintosingleusealiquotsandstoredat
-20oC.
3.PrepareserialdilutionsofD-lactatestandard(0to1mM):
3.1 Add10μLofD-lactatestocksolution(fromStep2)into990µL1xPBSbuffertogenerate1mMD-lactatestandardsolution.
Note:DilutedD-lactatestandardsolutionisunstable,andshouldbeusedwithin4hours.
3.2 Take200μLof1mMD-lactatestandardsolutiontoperform1:3serialdilutionstoget300,100,30,10,3,1and0μMserialdilutionsofD-lactatestandard.
3.3 AddserialdilutionsofD-lactatestandardandD-lactatecontainingtestsamplesintoawhiteclearbottom96-wellmicroplateasdescribedinTables1and2.
Table1LayoutofD-lactatestandardsandtestsamplesinawhiteclearbottom96-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=D-LactateStandards,BL=BlankControl,TS=TestSamples.
Table2Reagentcompositionforeachwell
D-LactateStandard | BlankControl | TestSample |
SerialDilutions*:50μL | DilutionBuffer:50μL | 50μL |
*Note:AddtheseriallydilutedD-Lactatestandardsfrom1μMto1mMintowellsfromLac1toLac7induplicate.
4.PrepareD-lactateassaymixture:
4.1 Add5mLofAssayBuffer(ComponentB)intoonebottleofEnzymeMix(ComponentA).
4.2 Add50µLNADstocksolution(100X,fromStep1)intothebottleofComponentA (fromStep4.1), and
mixwell.
Note:ThisD-lactateassaymixtureisenoughforone96-wellplate.Itisunstable,andshouldbeusedpromptlywithin2hoursandavoidexposuretolight.
Note2:Alternatively,onecanmakea50XofD-LactateEnzymeMixturestocksolutionbyadding100μLofH2OintothebottleofComponentA,andthenpreparetheD-lactateassaymixturebymixingthestocksolutionwithassaybuffer(ComponentB)and100xNADsolutionproportionally.Aliquotandstoretheunused50XD-LactateEnzymeMixturestocksolutionand100XNADsolutionat-20oC,andavoidfreeze-thawcycles.
5.RunD-lactateassay:
5.1 Add50μLofD-lactateassaymixture(fromStep4.2)toeachwellofD-lactatestandard,blankcontrol,andtestsamples(seeStep3.3)tomakethetotalD-lactateassayvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLofD-lactateassaymixtureintoeachwell.
5.2 Incubatethereactionatroomtemperaturefor30minutesto2hours,protectedfromlight.
5.3 MonitortheabsorbanceratioincreasewithanabsorbanceplatereaderatA575nm/A605nm.
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