| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 571/585 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | SmallMoleculeDetection DiagnosticMolecules |
| Related | BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1.Preparestocksolutions:
1.1250XAmplite™EthanolReagentstocksolution:Add40μLofDMSO(ComponentD)intothevialofAmplite™EthanolReagent(ComponentA).Thestocksolutionshouldbeusedpromptly.Anyremainingsolutionshouldbealiquotedandrefrozenat-20oC.
Note1:Avoidrepeatedfreeze-thawcycles.
Note2:TheAmplite™EthanolReagentisunstableinthepresenceofthiolssuchasdithiothreitol(DTT)and2-mercaptoethanol.ThefinalconcentrationofDTTor2-mercaptoethanolinthereactionshouldbenohigherthan10μM.TheAmplite™EthanolReagentisalsounstableathighpH(>8.5).Therefore,thereactionshouldbeperformedatpH7–8.Theprovidedassaybuffer(pH7.4)isrecommended.
1.2100XEthanolEnzymeMix:Add100μLofAssayBuffer(ComponentB)intothevialofEthanolEnzymeMix(ComponentC),andmixwell.
Note:TheunusedEthanolenzymemixsolutionshouldbedividedintosingleusealiquotsandstoredat20oC
2.Prepareassayreactionmixture:
Prepareassayreactionmixtureaccordingtothefollowingtables,protectedfromlight.
Table1Assayreactionmixtureforone96-wellplate
Components | Volume |
250XAmplite™ReagentStockSolution(fromStep1.1) | 20μL |
100XEthanolEnzymeMix(fromStep1.2) | 50μL |
AssayBuffer(ComponentB) | 5mL |
Totalvolume | 5.07mL |
Table2Layoutofethanolstandardsandtestsamplesinasolidblack96-wellmicroplate
BL | BL | TS | TS | …. | …. |
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ES1 | ES1 | …. | …. | …. | …. |
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ES2 | ES2 |
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ES3 | ES3 |
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ES4 | ES4 |
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ES5 | ES5 |
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ES6 | ES6 |
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ES7 | ES7 |
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Note:ES=EthanolStandards,BL=BlankControl,TS=TestSamples.
Table3Reagentcompositionforeachwell
EthanolStandard | BlankControl | TestSample |
SerialDilutions*:50μL | H2O:50μL | 50μL |
*Note1:AddtheserialdilutionsofEthanolstandardfrom0.0001%to0.1%intoeachwellfromES1toES7induplicate.
Note2:HighconcentrationofEthanol(e.g.,0.5%,finalconcentration)maycausereducedfluorescencesignalduetotheoveroxidationofAmplite™ethanolreagent(toanon-fluorescentproduct).
3.Runethanolassay:
3.1Prepareanethanolstandardbydilutingtheappropriateamountofthe100%ethanolstandard(ComponentE)intoH2Otoproduceethanolconcentrationrangingfrom0to0.1%,eachinavolumeof50μL.A0%ethanolcontrolisincludedasblankcontrol.Thefinalethanolconcentrationsshouldbetwofoldslower(i.e.,0to0.05%).
3.2Add50μLofassayreactionmixture(fromStep2)intoeachwellofethanolstandard,blankcontrol,andtestsamples(seeStep2,Table3)tomakethetotalethanolassayvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLofassayreactionmixtureintoeachwell.
3.3Incubatethereactionfor5to30minutesatroomtemperature,protectedfromlight.
3.4MonitorthefluorescenceintensitywithafluorescenceplatereaderatEx/Em=530-570nm/590-600nm(optimalEx/Em=540/590nm),cutoff570nm.
Note:Thecontentsoftheplatecanalsobetransferredtoawhiteclearbottomplateandreadbyanabsorbancemicroplatereaderatthewavelengthof576±5nm.Theabsorptiondetectionhaslowersensitivitycomparedtofluorescencereading.
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