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AAT Bioquest/Amplite™ Fluorimetric Acetylcholinesterase Assay Kit *Green Fluorescence*/11401/200 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 510/524 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | NeuroBIOLOGy NeurologicalEnzymes |
| Related | HydrolyticEnzymes MicroplateReaders |
| Spectrum | AdvancedSpectrumViewer |
1.Preparestocksolutions:
1.1 200XThiolite™Greenstocksolution:Add50µLofDMSO(ComponentE)intothevialofThiolite™Green(ComponentA)tomake200XThiolite™Greenstocksolution.
Note:TheunusedThiolite™Greenstocksolutionshouldbedividedintosingleusealiquots.Storeat-20oCandavoidexposuretolight.
1.2 500Xacetylthiocholinestocksolution:Add0.6mLofddH2Ointothevialofacetylthiocholine(ComponentC).
Note:Theunusedacetylthiocholinestocksolutionshouldbedividedintosingleusealiquotsandstoredat‑20oC.
1.3 Acetylcholinesterasestandardstocksolution:Add100µLofddH2Owith0.1%BSAintothevialofacetylcholinesterasestandard(ComponentD)tomakea50units/mLacetylcholinesterasestandardstocksolution.
Note:Theunusedacetylcholinesterasestandardstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.
2.Prepareacetylthiocholinereactionmixture:
Note:theacetylthiocholinereactionmixtureisnotstable,needbeusedwithin30min.
Preparetheacetylthiocholinereactionmixtureaccordingtothefollowingtableandkeepfromlight.
Table1.Acetylthiocholinereactionmixtureforone96-wellplate
Components | Volume |
Assaybuffer(ComponentB) | 5mL |
Thiolite™Greenstocksolution(200X,fromStep1.1) | 25µL |
Acetylthiocholinestocksolution(500X,fromStep1.2) | 10µL |
Totalvolume | 5.03mL |
3.Prepareseriallydilutedacetylcholinesterasestandards(0to100mU/mL):
3.1 Add20μLof50units/mLacetylcholinesterasestandardstocksolution(fromStep1.3)to980µLassaybuffer(ComponentC)togenerate1000mU/mLacetylcholinesterasestandardsolution.
Note:Dilutedacetylcholinesterasestandardsolutionisunstableandshouldbeusedwithin4hours.
3.2 Take200μLof1000mU/mLacetylcholinesterasestandardsolutiontoperform1:10and1:3serialdilutionstoget100,30,10,3,1,0.3,0.1and0mU/mLseriallydilutedacetylcholinesterasestandards.
3.3 Addseriallydilutedacetylcholinesterasestandardsand/oracetylcholinesterase-containingtestsamplesintoasolidblack96-wellmicroplateasdescribedinTables1and2.
Note:Treatcellsortissuesamplesasdesired.
Table1.Layoutofacetylcholinesterasestandardsandtestsamplesinasolidblack96-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.
Table2.Reagentcompositionforeachwell
AcetylcholinesteraseStandards | BlankControl | TestSample |
SerialDilutions*:50μL | AssayBuffer:50μL | 50μL |
*Note:Addtheseriallydilutedacetylcholinesterasestandardsfrom0.01to100mU/mLintowellsfromAS1toAS7induplicate.
4.Runacetylcholinesteraseassay:
4.1 Add50μLofacetylthiocholinereactionmixture(fromStep2.1)intoeachwelloftheacetylcholinesterasestandard,blankcontrol,andtestsamples(seeStep3.3)tomakethetotalacetylcholinesteraseassayvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLofacetylthiocholinereactionmixtureintoeachwell.
4.2 Incubatethereactionatroomtemperaturefor10to30minutes,protectedfromlight.
4.3 MonitorthefluorescenceincreasewithafluorescencemicroplatereaderatEx/Em=490/520nm.
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