| 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 |
| Spectrum | AdvancedSpectrumViewer |
1.Preparestocksolutions:
1.1 250XAmplite™Redstocksolution:Add40µLofDMSO(ComponentG)intothevialofAmplite™Red(ComponentA)tomake250XAmplite™Redstocksolution.
Note:TheunusedAmplite™Redstocksolutionshouldbedividedintosingleusealiquots.Storeat-20oCandavoidexposuretolight.
1.2 Acetylcholinesterasestandardstocksolution:Add100µLofAssayBuffer(ComponentE)intothevialofacetylcholinesterasestandard(ComponentD)tomakea50units/mLacetylcholinesterasestandardstocksolution.
Note:Theunusedacetylcholinesterasestandardstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.
1.3 Acetylcholinestocksolution:Add100µLofAssayBuffer(ComponentE)intothevialofAcetylcholine(ComponentC)tomake1000Xacetylcholinestocksolution.
Note:Theunusedacetylcholinestandardstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.
2.Prepareacetylcholinesteraseassaymixture:
2.1 Add5mLofAssayBuffer(ComponentE)tothebottleofAcetylcholinesteraseProbe(ComponentB)andmixwell.
2.2 Add5µLof1000Xacetylcholinestocksolution(fromStep1.3)intothebottleofAcetylcholinesteraseProbemixture(fromStep2.1)andmixwell.
2.3 Add20µLof250XAmplite™Redstocksolution(fromStep1.1)intothebottleofAcetylcholinesteraseProbemixture(fromStep2.2)tomaketheacetylcholinesteraseassaymixturebeforerunningtheassay.
Note:Theacetylcholinesteraseassaymixtureshouldbeusedpromptlyandkeptfromlight.Theassaybackgroundwouldincreasewithlongerstoragetime.
3.Prepareseriallydilutedacetylcholinesterasestandards(0to100mU/mL):
3.1 Add20μLof50units/mLacetylcholinesterasestandardstocksolution(fromStep1.2)to980μLDilution Buffer(ComponentF)togenerate1000mU/mLacetylcholinesterasestandardsolution.
Note:Dilutedacetylcholinesterasestandardsolutionisunstableandshouldbeusedwithin4hours.
3.2 Take200μLof1000mU/mLacetylcholinesterasestandardsolutiontoperform1:10and1:3serialdilutionstoget100,30,10,3,1,0.3,0.1and0mU/mLseriallydilutedacetylcholinesterasestandards.
3.3 Addseriallydilutedacetylcholinesterasestandardsand/oracetylcholinesterasecontainingtestsamplesintoasolidblack96-wellmicroplateasdescribedinTables1and2.
Note:Treatthecellsortissuesamplesasdesired.
Table1Layoutofacetylcholinesterasestandardsandtestsamplesinasolidblack96-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=AcetylcholinesteraseStandards;BL=BlankControl;TS=TestSamples
Table2Reagentcompositionforeachwell
AcetylcholinesteraseStandards | BlankControl | TestSample |
SerialDilutions*:50μL | DilutionBuffer:50μL | 50μL |
*Note:Addtheseriallydilutedacetylcholinesterasestandardsfrom0.01to100mU/mLintowellsfromAS1toAS7induplicate.
4.Runacetylcholinesteraseassay:
4.1 Add50μLofacetylcholinesteraseassaymixture(fromStep2.3)intoeachwellofacetylcholinesterasestandard,blankcontrol,andtestsamples(seeStep3.3)tomakethetotalacetylcholinesteraseassayvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLofacetylthiocholinereactionmixtureintoeachwell.
4.2 Incubatethereactionatroomtemperaturefor10to30minutes,protectedfromlight.
4.3 MonitorthefluorescenceincreasewithafluorescencemicroplatereaderatEx/Em=540/590nm.
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