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AAT Bioquest/Amplite™ Fluorimetric Glutathione Assay Kit *Green Fluorescence*/10055/200 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 510/524 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | SmallMoleculeDetection DiagnosticMolecules |
| Related | RedoxEnzymes BiochemicalAssays |
1.PrepareGSHstandardstocksolution:
Add200µLofAssayBuffer(ComponentB)intothevialofGSHStandard(ComponentC)tomake1mM(1nmol/µL)GSHstandardstocksolution.
Note:Theunused1mMGSHstandardstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.
2.Prepare100XThiolite™Greenstocksolution:
Add100µLofDMSO(ComponentD)intothevialofThiolite™Green(ComponentA)tomake100XThiolite™Greenstocksolution.
Note:TheunusedThiolite™Greenstocksolutionshouldbedividedintosingleusealiquots,storedat
-20 oC,andkeptfromlight.
3.PrepareGSHreactionmixture:
Add50µLof100XThiolite™Greenstocksolution(fromStep2)into5mLofAssayBuffer(ComponentB),andmixthemwell.
4.PrepareseriallydilutedGSHstandards(0to30μM):
4.1 Add30μLofGSHstandardstocksolution(fromStep1)into970µLofAssayBuffer(ComponentB)togenerate30µM(30pmol/µL)GSHstandardsolution.
Note:Diluted30µMGSHstandardsolutionisunstable.Usewithin4hours.
4.2 Take200μLof30μMGSHstandardsolutiontoperform1:3serialdilutionstoget10,3,1,0.3,0.1,0.03,0.01and0μMseriallydilutedGSHstandards.
4.3 AddGSHstandardsandGSH-containingorotherGSH-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
GSHStandards | BlankControl | TestSample |
SerialDilutions*:50μL | AssayBuffer:50μL | 50μL |
*Note:AddseriallydilutedGSHstandardsfrom0.01μMto10μMintowellsfromGS1toGS7induplicate.
5.RunGSHassay:
5.1 Add50μLofGSHreactionmixture(fromStep3)intoeachwellofGSHstandard,blankcontrol,andtestsamples(seeStep4.3)tomakethetotalGSHassayvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLofGSHreactionmixtureintoeachwell.
5.2 Incubatethereactionatroomtemperaturefor10minutesto1hour,protectedfromlight.
5.3 MonitorthefluorescenceincreaseatEx/Em=490/520nmusingafluorescenceplatereader.
| References&Citations |  CitationExplorer |
Comparisonofdifferentmethodstostudyeffectsofsilvernanoparticlesonthepro-andantioxidantstatusofhumankeratinocytesandfibroblasts
Authors:SebastianAhlberg,FiorenzaRancan,MatthiasEpple,KaterynaLoza,DavidHöppe,JürgenLademann,AnnikaVogt,BurkhardKleuser,ChristianGerecke,MartinaCMeinke
Journal:Methods(2016)
