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主营:研究并生产荧光和发光探针,信号转导研究的试剂
℡ 4000-520-616
℡ 4000-520-616
AAT Bioquest/Amplite™ Fluorimetric Total Thiol Quantitation Assay Kit *Green Fluorescence*/5524/200 Tests
产品编号:5524
市  场 价:¥56560.00
场      地:美国(厂家直采)
联系QQ:1570468124
电话号码:4000-520-616
邮      箱: info@ebiomall.com
美  元  价:$2828.00
品      牌: AAT Bioquest
公      司:AAT Bioquest
公司分类:
AAT Bioquest/Amplite™ Fluorimetric Total Thiol Quantitation Assay Kit *Green Fluorescence*/5524/200 Tests
商品介绍
Overview
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Ex/Em(nm)510/524
MWN/A
CAS#N/A
SolventN/A
StorageF/D/L
CategoryProteinBiochemistry
Generalproteins
RelatedBiochemicalAssays
Thedetectionandmeasurementoffreethiol(suchasfreecysteine,glutathioneandcysteineresiduesinproteins)isoneoftheessentialtasksforinvestigatingBIOLOGicalprocessesandeventsinmanybiologicalsystems.Themonitoringofreduced(GSH)andoxidizedglutathioneinbiologicalsamplesisessentialforevaluatingtheredoxanddetoxificationstatusofcellsandtissuesinrelationtotheprotectiveroleofglutathioneagainstoxidativeandfree-rADIcal-mediatedcellinjury.Disordersofcysteinemetabolismincludecystinosis,anautosomalrecessivediseaseproducedbyadefectinlysosomaltransport,andcystinuria,acommonheritabledisorderofaminoacidtransport.Therearefewreagentsorassaykitsavailableforquantitatingthiolsinbiologicalsystems.However,allthecommercialkitseitherlacksensitivityorhavetediousprotocols.OurAmplite™FluorimetricTotalThiolQutitationKitprovidesanultrasensitivefluorimetricassayforquantitatingthiolsthatexisteitherinasmallmolecule.Thekitusesaproprietarynon-fluorescentdyethatbecomesstronglyfluorescentuponreactingwiththiol.Thekitprovidesasensitive,one-stepfluorimetricmethodtodetectaslittleas1picomoleofcysteineorGSHina100µLassayvolume(10nMinconcentration).Theassayisrapidandrobust.Itcanbeperformedinaconvenient96-wellor384-wellmicrotiter-plateformatandeasilyadaptedtoautomationwithEx/Em=490nm/520nm.Forrapidquantifyingthiolgroupsinaprotein,werecommendyouuseourkits#5529and#5530thatareoptimizedforquantifyingproteinthiols.
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Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.PrepareGSHstandardstocksolution:

Add200µLofddH2OintotheGSHstandardvial(ComponentC)tomake1mM(1nmol/µL)stocksolution.

Note:TheunusedGSHsolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.

2.Prepare100XThiolite™Greenstocksolution:

Add100µLofDMSO(ComponentD)intothevialofThiolite™Green(ComponentA)tomake100Xstocksolution.

Note:TheunusedThiolite™Greensolutionshouldbedividedintosingleusealiquots,storedat-20oCandkeptfromlight.

3.PrepareGSHreactionmixture:

Add50µLof100XThiolite™Greenstocksolution(fromStep2)into5mLofassaybuffer(ComponentB),andmixwell.

Note1:ThisGSHassaymixture(GAM)isenoughforone96-wellplates.Itisunstableatroomtemperature,andshould 

  beusedpromptlywithin2hoursandavoidexposuretolight.

Note2:Alternatively,onecanmakeGSHAssayMixturebyadding100XThiolite™GreenstocksolutionwithAssay

 Bufferproportionally.

4.PrepareserialdilutionsofGSHstandard(0to10μM):

4.1   Add30μLofGSHstandardstocksolution(fromStep1)to970µLofassaybuffer(ComponentB)togenerate30µM(30pmol/µL)GSHstandard.

Note:DilutedGSHstandardsolutionisunstable.Usewithin4hours.

4.2   Take200μLof30μMGSHstandardsolutiontoperform1:3serialdilutionstoget10,3,1,0.3,0.1,0.03,0.01and0μMserialdilutionsofGSHstandard.

 

4.3   AddGSHstandardsandGSH-containingorotherthiol-containingtestsamplesintoasolidblack96-wellmicroplateasshowninTables1and2.

Note:Treatcellsortissuesamplesasdesired.

 

Table1LayoutofGSHstandardsandtestsamplesinasolidblack96-wellmicroplate

BL

BL

TS

TS

….

….

 

 

 

 

 

 

GS1

GS1

….

….

….

….

 

 

 

 

 

 

GS2

GS2

 

 

 

 

 

 

 

 

 

 

GS3

GS3

 

 

 

 

 

 

 

 

 

 

GS4

GS4

 

 

 

 

 

 

 

 

 

 

GS5

GS5

 

 

 

 

 

 

 

 

 

 

GS6

GS6

 

 

 

 

 

 

 

 

 

 

GS7

GS7

 

 

 

 

 

 

 

 

 

 

Note:GS=GSHStandards,BL=BlankControl,TS=TestSamples.

 

Table2Reagentcompositionforeachwell

GSHStandard

BlankControl

TestSample

SerialDilutions*:50μL

AssayBuffer:50μL

50μL

*Note:AddtheserialdilutionsofGSHstandardfrom0.01μMto10μMintowellsfromGS1toGS7induplicate.

5.RunGSHassay:

5.1   Add50μLofGSHreactionmixture(fromStep3.1)toeachwelloftheGSHstandard,blankcontrol,andtestsamples(seeStep4.3)tomakethetotalGSHassayvolumeof100µL/well.

Note:Fora384-wellplate,add25μLofsampleand25μLofGSHreactionmixtureintoeachwell.

5.2   Incubatethereactionatroomtemperaturefor10minutesto1hour,protectedfromlight.

5.3   MonitorthefluorescenceincreaseatEx/Em=490/520nmwithafluorescenceplatereader.

References&Citations
CitationExplorer

Activationoftranscriptionfactorsinhumanbronchialepithelialcellsexposedtoaqueousextractsofmainstreamcigarettesmokeinvitro
Authors:TakashiSekine,TadashiHirata,ToshikiMine,YasuoFukano
Journal:Toxicologymechanismsandmethods(2016):22--31


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