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AAT Bioquest/Amplite™ Fluorimetric L-Lactate Dehydrogenase (LDH) Assay Kit/13812/200 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-LDHstocksolution:
Add100µLofH2Oor1xPBSbufferintothevialofL-LDHstandard(ComponentD)tomake100U/mLL-LDHstandardsolution.
Note:TheunusedL-LDHstandardstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.
3.PrepareserialdilutionsofL-LDHstandard(0to300mU/mL):
3.1 Add10μLofL-LDHstocksolution(fromStep2)into990µL1xPBSbuffertogenerate1000mU/mLL-LDHstandardsolution.
Note:DilutedL-LDHstandardsolutionisunstable,andshouldbeusedwithin4hours.
3.2 Take200μLof1000mU/mlL-LDHstandardsolutiontoperform1:3serialdilutionstoget300,100,30,10,3,1,0.3and0
mU/mLserialdilutionsofL-LDHstandard.
3.3 AddserialdilutionsofL-LDHstandardandL-LDHcontainingtestsamplesintoasolidblack96-wellmicroplateasdescribedin
Tables1and2.
Table1LayoutofL-LDHstandardsandtestsamplesinasolidblack96-wellmicroplate
BL | BL | TS | TS | …. | …. |
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L-LDH1 | L-LDH1 | …. | …. | …. | …. |
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L-LDH2 | L-LDH2 |
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L-LDH3 | L-LDH3 |
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L-LDH4 | L-LDH4 |
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L-LDH5 | L-LDH5 |
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L-LDH6 | L-LDH6 |
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L-LDH7 | L-LDH7 |
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Note:L-LDH=L-LDHStandards,BL=BlankControl,TS=TestSamples.
Table2Reagentcompositionforeachwell
L-LDHStandard | BlankControl | TestSample |
SerialDilutions*:50μL | DilutionBuffer:50μL | 50μL |
*Note:AddtheseriallydilutedL-LDHstandardsfrom0.3mU/mLto300mU/mLintowellsfromL-LDH1toL-LDH7induplicate.
4.PrepareL-LDHassaymixture:
4.1 Add10mLofAssayBuffer(ComponentB)intothebottleofEnzymeProbe(ComponentA)tohaveenzymeprobemixture.
Note:Thisenzymeprobemixtureisenoughfortwo96-wellplate.Theunusedenzymeprobemixtureshouldbedividedintosingle
usealiquotsandstoredat-20oC.
4.2 Add50µLNADstocksolution(100X)into5mLenzymeprobemixture(fromStep4.1),andmixwell.
Note:ThisL-LDHassaymixtureisenoughforone96-wellplate.Itisnotstable,makeenoughforoneexperiment,anduseitpromptly.
5.RunL-LDHassay:
5.1 Add50μLofL-LDHassaymixture(fromStep4.2)toeachwellofL-LDHstandard,blankcontrol,andtestsamples(seeStep
3.3)tomakethetotalassayvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLassaymixtureintoeachwell.
5.2 Incubatethereactionatroomtemperaturefor30minutesto2hours,protectedfromlight.
5.3 MonitorthefluorescenceincreasewithafluorescenceplatereaderatEx/Em=530-570/590-600nm(optimalEx/Em=540/590
nm,cutoffat570nm).
Note:Thecontentsoftheplatecanalsobetransferredtoawhiteclearbottomplateandreadbyabsorbancemicroplatereader
attheratioofA575nm/A605nm.TheabsorptiondetectionhaslowersensitivitycomparedtofluorescencereADIng.
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