| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 410/None |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | NeuroBIOLOGy NeurologicalEnzymes |
| Related | HydrolyticEnzymes MicroplateReaders |
1.Preparestocksolutions:
1.1 20XDTNBstocksolution:Add0.6mLofAssayBuffer(ComponentB)intothevialofDTNB(ComponentA)tomake20XDTNBstocksolution.
Note:TheunusedDTNBstocksolutionshouldbedividedintosingleusealiquots.Storeat-20oCandkeepfromlight.
1.2 20XAcetylthiocholinestocksolution:Add0.6mLofddH2Ointothevialofacetylthiocholine(Component C).
Note:Theunused20Xacetylthiocholinestocksolutionshouldbedividedintosingleusealiquotsandstoredat‑20oC.
1.3 Acetylcholinesterasestocksolution:Add100µLofddH2Owith0.1%BSAintothevialofacetylcholinesterasestandard(ComponentD)tomakea50units/mLacetylcholinesterasestocksolution.
Note:Theunusedacetylcholinesterasestocksolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.
2.Prepareacetylthiocholinereactionmixture:
PreparetheacetylthiocholinereactionmixtureaccordingtoTable1andkeepfromlight.
Table1Acetylthiocholinereactionmixtureforone96-wellplate
Components | Volume |
AssayBuffer(ComponentB) | 4.5mL |
20XDTNBStockSolution(fromStep1.1) | 250µL |
20XAcetylthiocholineStocksolution(fromStep1.2) | 250µL |
Totalvolume | 5mL |
3.Prepareserialdilutionsofacetylcholinesterasestandard(0to1000mU/mL):
3.1 Add20μLof50units/mLacetylcholinesterasestocksolution(fromStep1.3)to980µLofassaybuffer(ComponentB)togenerate1000mU/mLacetylcholinesterasestandardsolution.
Note:Dilutedacetylcholinesterasestandardsolutionisunstableandshouldbeusedwithin4hours.
3.2 Take200μLof1000mU/mLacetylcholinesterasestandardtoperform1:3serialdilutionstoget300,100,30,10,3,1and0mU/mLserialdilutionsofacetylcholinesterasestandard.
3.3 Addserialdilutionsofacetylcholinesterasestandardandacetylcholinesterase-containingtestsamplesintoawhite/clearbottom96-wellmicroplateasdescribedinTables2and3.
Note:Treatcellsortissuesamplesasdesired.
Table2.Layoutofacetylcholinesterasestandardsandtestsamplesinawhite/clearbottom96-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
Table3.Reagentcompositionforeachwell
AcetylcholinesteraseStandard | BlankControl | TestSample |
SerialDilutions*:50μL | AssayBuffer:50μL | 50μL |
*Note:Addtheserialdilutionsofacetylcholinesterasestandardfrom1to1000mU/mLintowellsfromAS1toAS7induplicate.
4.Runacetylcholinesteraseassay:
4.1 Add50μLofacetylthiocholinereactionmixture(fromStep2.1)toeachwelloftheacetylcholinesterasestandard,blankcontrol,andtestsamples(seeStep3.3)tomakethetotalacetylcholinesteraseassayvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLofacetylthiocholinereactionmixtureineachwell.
4.2 Incubatethereactionfor10to30minutesatroomtemperature,protectedfromlight.
4.3 Monitortheabsorbanceincreasewithanabsorbancemicroplatereaderat410±5nm.
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