| 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:
1.1 Amplite™Redstocksolution(250X):Add40µLofDMSO(ComponentE)intothevialofAmplite™Red(ComponentA)tomakea250Xstocksolution.
Note:TheunusedAmplite™Redstocksolutionshouldbedividedintosingleusealiquots.Storeat-20oCandavoidexposuretolight.
1.2 Cholinestocksolution:Add400µLofddH2OintothevialofCholineStandard(ComponentC)tomake50mMcholinestocksolution.
Note:Theunusedcholinestocksolutionshouldbedividedintosingleusealiquotsandstoredat‑20 oC.
2.Preparecholineassaymixture:
2.1 Add5mLofAssayBuffer(ComponentD)tothebottleofCholineProbe(ComponentB)andmixwell.
2.2 Add20µLofAmplite™Redstocksolution(250X,fromStep1.1)intotheCholineProbebottle(fromStep2.1)tomakethecholineassaymixture.
Note:Thecholineassaymixtureshouldbeusedpromptlyandkeptfromlight.Theassaybackgroundwouldincreasewithlongerstoragetime.
3.Prepareserialdilutionsofcholinestandard(0to30μM):
3.1 Add20μLof50mMcholinestandardstocksolution(fromStep1.2)to980µLAssayBuffer(ComponentD)togenerate1000μMstandardsolution.
Note:Dilutedcholinestandardsolutionisunstable,andshouldbeusedwithin4hours.
3.2 Take30μLof1000μMstandard(fromStep3.1)to970µLAssayBuffer(ComponentD)togenerate30μMcholinestandardsolution,andthenperform1:3serialdilutionstoget10,3,1,0.3,0.1,0.03,and0μMcholinestandard.
3.3 Addcholinestandardsandcholinecontainingtestsamplesintoa96-wellsolidblackmicroplateasdescribedinTables1and2.
Note:Treatthecellsortissuesamplesasdesired.
Table1.Layoutofcholinestandardsandtestsamplesinasolidblack96-wellmicroplate*
BL | BL | TS | TS | …. | …. |
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CS1 | CS1 | …. | …. | …. | …. |
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CS2 | CS2 |
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CS3 | CS3 |
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CS4 | CS4 |
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CS5 | CS5 |
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CS6 | CS6 |
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CS7 | CS7 |
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*Note:CS=CholineStandards;BL=BlankControl;TS=TestSamples
Table2.Reagentcompositionforeachwell*
CholineStandard | BlankControl | TestSample |
Serialdilutions*(50μL) | Assaybuffer:50μL | 50μL |
*Note:Addtheseriallydilutionsofcholinestandardsfrom0.03to30μMintowellsfromCS1toCS7induplicate.
4.Runcholineassay:
4.1 Add50μLofcholineassaymixture(fromStep2.2)toeachwellofthecholinestandard,blankcontrol,andtestsamples(seeStep3.3)tomakethetotalcholineassayvolumeof100µL/well.
Note:Fora384-wellplate,add25μLsampleand25μLofcholineassaymixtureperwell.
4.2 Incubatethereactionatroomtemperaturefor10to30minutes,protectedfromlight.
4.3 MonitorthefluorescenceincreasewithafluorescencemicroplatereaderatEx/Em=540/590nm(cutoffat570nm).
| References&Citations |  CitationExplorer |
StructuralsnapshotsofthecatalyticcycleofthephosphodiesteraseAutotaxin
Authors:JensHausmann,Willem-JanKeune,AgnesLHipgraveEderveen,LeonievanZeijl,RobbiePJoosten,AnastassisPerrakis
Journal:JournalofstructuralBIOLOGy(2016)
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Besidesthestandardcatalogproductswealsooffercustomservicetomeetyourspecialresearchneeds.Ourcurrentservicesincludecustomsynthesisofcolorimetric,fluorescentandluminescentprobes,customdevelopmentofbiochemical,cell-basedanddiagnosticassaysandcustomscreeningofyourcompoundlibrariesagainstyourdefinedtargetsusingourvalidatedHTSassays.
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