| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 571/585 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | NeuroBIOLOGy NeurologicalEnzymes |
| Related | DiagnosticMolecules MicroplateReaders BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1.Preparestocksolutions:
1.1 250XAmplite™Redstocksolution:Add40µLofDMSO(ComponentE)intothevialofAmpliteRed™(ComponentA)tomakea250XAmplite™Redstocksolution.
Note:TheunusedAmpliteRed™stocksolutionshouldbedividedintosingleusealiquots.Storeat-20oCandavoidexposuretolight.
1.2 Acetylcholinestocksolution:Add200µLofddH2OintothevialofAcetylcholineStandard(ComponentC)tomake50mMacetylcholinestocksolution.
Note:Theunusedacetylcholinestocksolutionshouldbedividedintosingleusealiquotsandstoredat‑20 oC.
2.Prepareacetylcholineassaymixture:
2.1 Add5mLofAssayBuffer(ComponentD)tothebottleofAcetylcholineProbe(ComponentB)andmixwell.
2.2 Add20µLof250XAmpliteRed™stocksolution(fromStep1.1)intothebottleofAcetylcholineProbesolution(fromStep2.1)tomaketheacetylcholineassaymixture.
Note:TheAssaymixtureshouldbeusedpromptlyandkeptfromlight.Theassaybackgroundwouldincreasewithlongerstoragetime.
3.Prepareserialdilutionsofacetylcholinestandard(0to100μM):
3.1 Add20μLof50mMacetylcholinestandardstocksolution(fromStep1.2)to980µLAssayBuffer(ComponentD)togenerate1000μMacetylcholinestandardsolution.
Note:Dilutedacetylcholinestandardsolutionisunstable,andshouldbeusedwithin4hours.
3.2 Take200μLof1000μMacetylcholinestandardtoperform1:10and1:3serialdilutionstoget100,30,10,3,1,0.3,0.1and0μMserialdilutionsofacetylcholinestandard.
3.3 Addacetylcholinestandardsandacetylcholinecontainingtestsamplesintoa96-wellsolidblackmicroplateasdescribedinTables1and2.
Note:Treatcellsortissuesamplesasdesired.
Table1Layoutofacetylcholinestandardsandtestsamplesinasolidblack96-wellmicroplate
BL | BL | TS | TS | …. | …. |
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AS1 | AS1 | …. | …. | …. | …. |
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AS2 | AS2 |
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AS3 | AS3 |
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AS4 | AS4 |
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AS5 | AS5 |
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AS6 | AS6 |
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AS7 | AS7 |
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Note:AS=AcetylcholineStandards;BL=BlankControl;TS=TestSamples
Table2Reagentcompositionforeachwell
AcetylcholineStandard | BlankControl | TestSample |
SerialDilutions*:50μL | AssayBuffer:50μL | 50μL |
*Note:Addtheserialdilutionsofacetylcholinestandardfrom0.01to100μMintowellsfromAS1toAS7induplicate.
4.Runacetylcholineassay:
4.1 Add50μLofacetylcholineassaymixture(fromStep2.2)toeachwelloftheacetylcholinestandard,blankcontrol,andtestsamples(seeStep3.3)tomakethetotalacetylcholineassayvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLofacetylcholineassaymixtureperwell.
4.2 Incubatethereactionatroomtemperaturefor10to30minutes,protectedfromlight.
4.3 MonitorthefluorescenceincreasewithafluorescencemicroplatereaderatEx/Em=540/590nm.
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