| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 550/None |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | SmallMoleculeDetection DiagnosticMolecules |
| Related | RedoxEnzymes BiochemicalAssays |
1.Prepare2XAldeView™Yellowreactionmixture:
Add5mLofAssaySolution(ComponentB)intothebottleofAldeView™Yellow(ComponentA),andmixwell.
Note1:5mLofthe2XAldeView™Yellowreactionmixtureisenoughfor1plate.Thereactionmixtureisnotstable.Usewithin2hours.
Note2:Assaysolution(ComponentB)ispotentiallyhazardous.Weargloveswhenhandlingit.
2.Prepareserialdilutionsofaldehydestandard(0to1mM):
2.1 Add1mLofDilutionBuffer(ComponentD)intothevialofAldehydeStandard(ComponentC)tomakea10mMaldehydestandardstocksolution.
Note:Theunused10mMAldehydestandardstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.
2.2 Take100μLof10mMaldehydestandardstocksolution(fromStep2.1)toperform1:10,and1:3serialdilutionstoget1000,300,100,30,10,3,1,0.3,and0μMserialdilutionsofaldehydestandard.
2.3 Addserialdilutionsofaldehydestandardandaldehyde-containingtestsamplesintoa96-wellwhite/clearbottommicroplateasdescribedinTables1and2.
Note1:BothBSAandTween20willinterferetheassay,uselessthan0.001%BSAand0.01%Tween20inthesamples.
Note2:Ifthealdehyde-containingsamplesarefromtheenzymereactionsuchasfructose-1,6-bisphosphatewithfructose-1,6-bisphosphatealdolase,prepare50µLofenzymereaction(25µLfora384-wellplate)asdesired.Incubatetheenzymereactionat37oCforatleast1hour.Thecomponentsofenzymereactionshouldbeoptimizedasneeded(e.g.anoptimizedbuffersystemmightberequiredforaspecificenzymereaction).
Note3:Inmostcases,DilutionBuffer(ComponentD)canalsobeusedforrunningenzymereactionifyoudonothaveanoptimizedenzymebuffer.
Table1LayoutofAldehydestandardsandtestsamplesinawhite/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=AldehydeStandards,BL=BlankControl,TS=TestSamples.
Table2Reagentcompositionforeachwell
AldehydeStandard | BlankControl | TestSample |
SerialDilutions*:50μL | Assaybuffer:50μL | 50μL |
*Note:Addtheserialdilutionsofaldehydestandardfrom0.3μMto1000μMintowellsfromAS1toAS7induplicate.
3.Runaldehydeassay:
3.1 Add50μLof2XAldeView™Yellowreactionmixtures(fromStep1)intoeachwellofthealdehydestandard,blankcontrol,andtestsamples(seeStep2.3)tomakethetotalaldehydeassayvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLofaldehydereactionmixtureintoeachwell.
3.2 Incubatethereactionmixtureatroomtemperaturefor30to60minutes,protectedfromlight.
3.3 Monitortheabsorbanceincreasewithanabsorbanceplatereaderat405or550nm.
Note:DifferentconcentrationsofthealdehydemightformdifferentcolorswithAldeView™Yellow.Atlowerconcentration,theabsorbanceat405nmgivesthebestresult.However,athigherconcentration,theabsorbancetendstoshiftto550nm.
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