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AAT Bioquest/Amplite™ Rapid Fluorimetric Total Thiol Quantitation Assay Kit *Green Fluorescence*/5528/200 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 510/524 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | ProteinBiochemistry Generalproteins |
| Related | BiochemicalAssays |
1.PrepareGSHstandardstocksolution:
Add200µLofddH2OintothevialofGSHStandard(ComponentC)tomake1mM(1nmol/µL)stocksolution.
Note:TheunusedGSHsolutionshouldbedividedintosingleusealiquotsandstoredat-20ºC.
2.Prepare100XThiolite™Green520WSstocksolution:
Add100µLofddH2OintothevialofThiolite™Green520WS(ComponentA)tomake100Xstocksolution.
Note:TheunusedThiolite™Green520WSsolutionshouldbedividedintosingleusealiquots,storedat-20ºCandkeptfromlight.
3.PrepareGSHreactionmixture:
Add50µLof100XThiolite™Green520WSstocksolution(fromStep2)into5mLofAssayBuffer(ComponentB),andmixwell.
Note1:ThisGSHreactionmixtureisenoughfortwo96-wellplates.Itisstableat4ºCfor6hourswhenkeptfromlight.
Note2:Alternatively,onecanmakeGSHreactionmixturebyadding100XThiolite™Green520WSstocksolutionwithAssayBufferproportionally.
4.PrepareserialdilutionsofGSHstandard(0to10μM):
4.1 Add30μLofGSHstandardstocksolution(fromStep1)to970µLofAssayBuffer(ComponentB)togenerate30µM(30pmol/µL)GSHstandard.
Note:DilutedGSHstandardsolutionisunstable.Usewithin4hours.
4.2 Take200μLof30μMGSHstandardsolutiontoperform1:3serialdilutionstogetapproximately10,3,1,0.3,0.1,0.03,0.01and0μMserialdilutionsofGSHstandard.
4.3 AddGSHstandardsandGSH-containingorotherthiol-containingtestsamplesintoasolidblack96-wellmicroplateasshowninTables1and2.
Note:Treatcellsortissuesamplesasdesired.
Table1LayoutofGSHstandardsandtestsamplesinasolidblack96-wellmicroplate
BL | BL | TS | TS | …. | …. |
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GS1 | GS1 | …. | …. | …. | …. |
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GS2 | GS2 |
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GS3 | GS3 |
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GS4 | GS4 |
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GS5 | GS5 |
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GS6 | GS6 |
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GS7 | GS7 |
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Note:GS=GSHStandards,BL=BlankControl,TS=TestSamples.
Table2Reagentcompositionforeachwell
GSHStandard | BlankControl | TestSample |
SerialDilutions*:50μL | AssayBuffer:50μL | 50μL |
*Note:AddtheserialdilutionsofGSHstandardfrom0.01μMto10μMintowellsfromGS1toGS7induplicate.
5.RunGSHassay:
5.1 Add50μLofGSHreactionmixture(fromStep3)toeachwelloftheGSHstandard,blankcontrol,andtestsamples(seeStep4.3)tomakethetotalGSHassayvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLofGSHreactionmixtureintoeachwell.
5.2 Incubatethereactionatroomtemperaturefor10minutesto1hour,protectedfromlight.
5.3 MonitorthefluorescenceincreaseatEx/Em=490/525nm(cutoff:510nm)withafluorescenceplatereader.
| References&Citations |  CitationExplorer |
Activationoftranscriptionfactorsinhumanbronchialepithelialcellsexposedtoaqueousextractsofmainstreamcigarettesmokeinvitro
Authors:TakashiSekine,TadashiHirata,ToshikiMine,YasuoFukano
Journal:Toxicologymechanismsandmethods(2016):22--31
