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AAT Bioquest/Amplite™ Fluorimetric Glutathione GSH/GSSG Ratio Assay Kit *Green Fluorescence*/10056/200 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:TheunusedGSHstandardstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.
2.PrepareGSSGstandardstocksolution:
Add200µLofddH2OintothevialofGSSGStandard(ComponentF)tomake1mM(1nmol/µL)GSSGstandardstocksolution.
Note:TheunusedGSSGstandardstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.
3.Prepare100XThiolite™Greenstocksolution:
Add100µLofDMSO(ComponentD)intothevialofThiolite™Green(ComponentA)tomake100XThiolite™Greenstocksolution.
Note:TheunusedThiolite™Greenstocksolutionshouldbedividedintosingleusealiquots,storedat-20 oC,andavoidlight.
4.PrepareGSHAssayMixture(GAM):
Add100µLof100XThiolite™Greenstocksolution(fromStep3)into10mLofAssayBuffer(ComponentB),andmixthemwellbyvortexing.
Note1:ThisGSHassaymixture(GAM)isenoughfortwo96-wellplates.Itisunstableatroomtemperature,andshouldbeusedpromptlywithin2hoursandavoidexposuretolight.
Note2:Alternatively,onecanmakeGSHAssayMixturebyadding100XThiolite™GreenstocksolutionwithAssayBufferproportionally.
5.PrepareTotalGSHAssayMixture(TGAM):
Add5mLofGAM(fromStep4)intothebottleofGSSGProbe(ComponentE),andmixthemwell.
Note1:ThisTotalGSHassaymixture(TGAM)isenoughforone96-wellplates.Itisunstableatroomtemperature,andshouldbeusedpromptlywithin2hoursandavoidexposuretolight.
Note2:Alternatively,onecanmakea25XGSSGProbebyadding200μLofddH2OintothebottleofComponentE,andthenpreparetheTGAMassaymixturebymixthestocksolutionwithGAM(fromStep4)proportionally. Aliquotandstoretheunused25XGSSGprobestocksolutionat-20oC,andavoidfreeze-thawcycles.
6.PrepareseriallydilutedGSHstandards(0to5μM):
6.1 Add10μLofGSHstandardstocksolution(fromStep1)to990µLofAssayBuffer(ComponentB)togenerate10µM(10pmol/µL)GSHstandardsolution.
Note:DilutedGSHstandardsolutionisunstable.Usewithin4hours.
6.2 Take200μLof10μMGSHstandardsolutiontoperform1:2serialdilutionstoget5,2.5,1.25,0.625,0.3125,0.1563,0.0781,and0μMseriallydilutedGSHstandards.
6.3 AddGSHstandardsandGSH-containingtestsamplesintoasolidblack96-wellmicroplateasshowninTable1andTable2.WhenjustGSHassayisneeded,fillONLYthewellsintwoleftcolumns(PanelA)accordingtoTable1.SkipStep7andgodirectlytoStep8.
Note:Treatcellsortissuesamplesasdesired.
7.PrepareseriallydilutedGSSGstandards(0to5μM):
7.1 Add10μLofGSSGstandardstocksolution(fromStep2)into990µLofAssayBuffer(ComponentB)togenerate10µM(10pmol/µL)GSSGstandardsolution.
Note:DilutedGSSGstandardsolutionisunstable.Usewithin4hours.
7.2 Take200μLof10μMGSSGstandardsolutiontoperform1:2serialdilutionstoget5,2.5,1.25,0.625,0.3125,0.1563,0.0781,and0μMseriallydilutedGSSGstandards.TheconcentrationsofTotalGSHstandardsolutionsshouldbetwicetheconcentrationsofGSSGstandardsolutionsas10,5,2.5,1.25,0.625,0.3125,0.1563,and0μM.
7.3 AddGSSGstandardsandGSH-containingtestsamplesintoasolidblack96-wellmicroplateasshowninTables1and2.WhenTotalGSHassayisneeded,fillthewellsinbothPanelA(left)andPanelB(right)accordingtoTable1.
Note:Treatcellsortissuesamplesasdesired.
Table1LayoutofGSHstandards,GSSGstandards,andtestsamplesinasolidblack96-wellmicroplate
PanelA(GSH) PanelB(GSSG)
GSH1 | GSH1 | TS…. | TS…. |
|
| GSSG1 | GSSG1 | TS…. | TS…. |
|
|
GSH2 | GSH2 | …. | …. |
|
| GSSG2 | GSSG2 | …. | …. |
|
|
GSH3 | GSH3 |
|
|
|
| GSSG3 | GSSG3 |
|
|
|
|
GSH4 | GSH4 | BL |
|
|
| GSSG4 | GSSG4 |
|
|
|
|
GSH5 | GSH5 |
|
|
|
| GSSG5 | GSSG5 |
|
|
|
|
GSH6 | GSH6 |
|
|
|
| GSSG6 | GSSG6 |
|
|
|
|
GSH7 | GSH7 |
|
|
|
| GSSG7 | GSSG7 |
|
|
|
|
BL | BL |
|
|
|
| BL | BL |
|
|
|
|
Note:GSH=GSHStandards,GSSG=GSSGStandards,BL=BlankControl,TS=TestSamples.
Table2Reagentcompositionforwells
GSHand/orGSSGStandards | BlankControl | TestSample |
SerialDilutions*:50μL | AssayBuffer:50μL | 50μL |
*Note:AddseriallydilutedGSHstandardsfrom0.01μMto5μMintowellsfromGS1toGS7induplicate.AddtheseriallydilutedGSSGstandardfrom0.01μMto5μMintowellsfromGSSG1toGSSG7induplicate.AddTSintowellsinbothPanelAandPanelB.
8.RunGSHandTotalGSHassay:
8.1 Add50μLofGSHAssayMixture(GAM,fromStep4)intothewellsinPanelA(left)ofGSHstandard,blankcontrol,andtestsamples(fromStep6.3)tomakethetotalassayvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLofGSHAssaymixtureintoeachwell.
8.2 IftotalGSH(inreducedandoxidizedstates)assayisneeded,add50μLofTotalGSHAssayMixture(TGAMfromStep5)intothewellsinPanelB(right)ofGSSGstandard,blankcontrol,andtestsamples(FromStep6.3)tomakethetotalassayvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLofTotalGSHAssayMixtureintoeachwell.
8.3 Incubatethereactionatroomtemperaturefor10minutesto1hour,protectedfromlight.
8.4 MonitorthefluorescenceincreaseatEx/Em=490/520nmwithafluorescenceplatereader.
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