| 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.PrepareD-LDHstocksolution:
Add100µLofH2Oor1xPBSbufferintothevialofD-LDHstandard(ComponentD)tomake100U/mLD-LDHstandardsolution.
Note:TheunusedD-LDHstandardstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.
3.PrepareserialdilutionsofD-LDHstandard(0to300mU/mL):
3.1 Add10μLofD-LDHstocksolution(fromStep2)into990µL1xPBSbuffertogenerate1000mU/mLD-LDHstandardsolution.
Note:DilutedD-LDHstandardsolutionisunstable,andshouldbeusedwithin4hours.
3.2 Take200μLof1000mU/mlD-LDHstandardsolutiontoperform1:3serialdilutionstoget300,100,30,10,3,1,0.3and0mU/mLserialdilutionsofD-LDHstandard.
3.3 AddserialdilutionsofD-LDHstandardandD-LDHcontainingtestsamplesintoasolidblack96-wellmicroplateasdescribedinTables1and2.
Table1LayoutofD-LDHstandardsandtestsamplesinasolidblack96-wellmicroplate
BL | BL | TS | TS | …. | …. |
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D-LDH1 | D-LDH1 | …. | …. | …. | …. |
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D-LDH2 | D-LDH2 |
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D-LDH3 | D-LDH3 |
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D-LDH4 | D-LDH4 |
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D-LDH5 | D-LDH5 |
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D-LDH6 | D-LDH6 |
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D-LDH7 | D-LDH7 |
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Note:D-LDH=D-LDHStandards,BL=BlankControl,TS=TestSamples.
Table2Reagentcompositionforeachwell
D-LDHStandard | BlankControl | TestSample |
SerialDilutions*:50μL | DilutionBuffer:50μL | 50μL |
*Note:AddtheseriallydilutedD-LDHstandardsfrom0.3mU/mLto300mU/mLintowellsfromD-LDH1toD-LDH7induplicate.
4.PrepareD-LDHassaymixture:
4.1 Add10mLofAssayBuffer(ComponentB)intothebottleofEnzymeProbe(ComponentA)tohave
enzymeprobemixture.
Note:Thisenzymeprobemixtureisenoughfortwo96-wellplate.Theunusedenzymeprobemixtureshouldbedividedintosingleusealiquotsandstoredat-20oC.
4.2 Add50µLNADstocksolution(100X)into5mLenzymeprobemixture(fromStep4.1),andmixwell.
Note:ThisD-LDHassaymixtureisenoughforone96-wellplate.Itisnotstable,makeenoughforoneexperiment,anduseitpromptly.
5.RunD-LDHassay:
5.1 Add50μLofD-LDH assaymixture(fromStep4.2)toeachwellofD-LDH standard,blankcontrol,andtestsamples(seeStep3.3)tomakethetotalassayvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLassaymixtureintoeachwell.
5.2 Incubatethereactionatroomtemperaturefor30minutesto2hours,protectedfromlight.
5.3 MonitorthefluorescenceincreasewithafluorescenceplatereaderatEx/Em=530-570/590-600nm(optimalEx/Em=540/590nm,cutoffat570nm).
Note:ThecontentsoftheplatecanalsobetransferredtoawhiteclearbottomplateandreadbyabsorbancemicroplatereaderattheratioofA575nm/A605nm.TheabsorptiondetectionhaslowersensitivitycomparedtofluorescencereADIng.
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