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AntibodiesOnline/Thiol Fluorescent Detection Kit/ABIN577645/96 tests
Antigen
Thiol
MinimumDetectionLimit
< 0.5 pM
Application
BiochemicalAssay(BCA)
Options
Bulkdiscount
Supplier
SupplierProductNo.
PurposeTheDetectX®ThiolkitisdesignedtoquantitativelymeasurethiolgroupsgeneratedorpresentinBIOLOGicalsamples.
BrandDetectX®
SampleTypeBiologicalBuffers
DetectionMethodFluorometric
SpecificitySampleTypesvalidated:ProteinsandPeptidesinBiologicalBuffers
Sensitivity4.62nM
CharacteristicsTheThiolFluorescentDetectionkitallowsuserstoaccuratelydeterminetheextentoffreethiolcontentinsamplesusingaproprietarysubstrate,ThioStar®,thatisconvertedtoabrightlyfluorescentproductuponreactionwiththiolsinthesample.Thefluorescentsignalisreadat510nmwithexcitationat410nminafluorescentplatereader.Thethiolinthesamplecaneitherbeonethatisgeneratedbyareaction,suchastheendproductofanenzymaticreactionthatproducesreducedglutathione,orcanbethecysteinecontentoftheproteintobemeasured.Thegraphshowsthemeasurementofthiolcontentinasamplecontainingupto4MguanidineHCl,equivalentto8MGuHClinthesample.Freethiolsinbiologicalsystemshaveimportantroles.Oxidativelymodifiedthiolgroupsofcysteineresiduesareknowntomodulatetheactivityofagrowingnumberofproteins.Oneofthemostpressingproblemswiththisapproachistoaccuratelydeterminetheextentofmodificationofspecificaminoacids,suchascysteineresidues,inacomplexproteinsample.
ComponentsBlackMicrotiterPlate
MaterialnotincludedDeionizedordistilledwater.
Repeaterpipetwithdisposabletipscapableofdispensing25μL.
Polypropylenedisposabletesttubesformakingdilutions.
Fluorescence96wellplatereadercapableofreADIngfluorescentemissionat510nm,withexcitationat390nm.
Pleasecontactyourplatereadermanufacturerforsuitablefiltersets.
Setplateparametersfora96-wellCorningCostar3686plate.
See:www.arborassays.com/resources/#general-infoforplatedimensiondata.
Softwareforconvertingrawrelativefluorescentunit(FLU)readingsfromtheplatereaderandcarryingoutfourparameterlogisticcurve(4PLC)fitting.
BackgroundFreethiolsinbiologicalsystemshaveimportantroles.Oxidativelymodifiedthiolgroupsofcysteineresiduesareknowntomodulatetheactivityofagrowingnumberofproteins.Oneofthemostpressingproblemswiththisapproachistoaccuratelydeterminetheextentofmodificationofspecificaminoacids,suchascysteineresidues,inacomplexproteinsample,especiallyinthepresenceofchaotropicagentssuchasguandinehydrochloride.TheDetectX®kitallowsuserstoaccuratelydeterminetheextentoffreethiolcontentinsamplesusingaproprietarynon-fluorescentsubstrate,ThioStar®,thatisconvertedtoabrightlyfluorescentproductuponreactionwiththethiolinthesample.Thethiolinthesamplecaneitherbeonethatisgeneratedbyareaction,suchastheendproductofanenzymaticreactionsuchasglutathione,orcanbethecysteinecontentoftheproteintobemeasured.Thisassayhasbeentestedwithsamplesinguanidinehydrochlorideconcentrationsupto2MintheAssayBuffersuppliedinthekit.Thisallowsthethiolcontentofunfoldedproteinstobeaccuratelydetermined.Althoughwehaveprovidedacysteinederivativeasastandardthatcanbeusedtoquantifyfreecysteinesonpeptidesandproteins,wesuggestthattheassaybecalibratedtoastandardthatchemicallyisascloseaspossIBLetothethiolbeingmeasured.Forexample,iftheenduserismeasuringglutathionewiththekit,thentheassayshouldbecalibratedtoaknown,validatedglutathionestandardpreparation.®www.ArborAssays.com3
ApplicationNotesThisassayhasbeenvalidatedforsamplesinanumberofbiologicalbuffersincludingTris,phosphate,andcitrateat pHsclosetoneutrality.
Allsamplesshouldbedilutedatleast1:10intheAssayBufferpriortoanalyzing.
Allsamplesandbuffersshouldbefreeofexcessthiolsandreducingagentssuchasß-mercaptoethanol,TCEP,orDTT.
Thisassayhasbeenvalidatedforsamplesinguanidinehydrochloridesolutionsupto4MwhenthesesamplesaredilutedwithanequalvolumeofAssayBuffer.
TheeffectofGuHClconcentrationisshownbelow.1201008060402004M2M1M0.(5=((M8==M4M2M(=sss1Mamamamsplppame)lel)e)ple)MolarityofguanidinehClinWell
Plate96wells
ProtocolAstandardisprovidedtogenerateastandardcurvefortheassayandallsamplesshouldbereadoffthecurvegenerated.
SamplesandstandardsarePipettedintoablackmicrotiterplate.
AftermixingthesampleorstandardwithThioStarandincubatingatroomtemperaturefora30minuteincubation,thefluorescentproductisreadat510nminafluorescentplatereaderwithexcitationat390nm.
Theconcentrationofthethiolinthesampleiscalculated,aftermakingasuitablecorrectionforanydilutionofthesample,usingsoftwareavailablewithmostfluorescenceplatereaders.
Wehaveprovideda96wellplateformeasurementbutthisassayisadaptableformeasurementinhigherdensityplateformats.
Theendusershouldensurethattheirblackplateissuitableforusewiththesereagentspriortorunningsamples.
Sincebiologicallygeneratedfreethiols,suchasglutathione,andproteinthiolgroups,existindifferentenvironmentswesuggestthattheendusercalibratetheamountofthiolpresentorgeneratedusingasuitablestandard.
ReagentPreparation

Allowthekitreagentstocometoroomtemperaturefor30minutes.
Ensurethatallsampleshavereachedroomtemperatureandhavebeendilutedasappropriatepriortorunningtheminthekit.
BufferPreparationTheAssayBufferConcentrateshouldbediluted1:2bytakingonepartoftheConcentrateandaddingonepartofdeionizedwaterpriortouse.
Itisstableforupto3monthswhenstoredat4°C.thiostar®thiolDetectionreagentAllowtheziplocpouchtowarmcompletelytoroomtemperaturepriortoopening.
RemovethevialofThioStarReagent.
Add1.5mLoftheprovidedDMSOtothevial.
Vortexthoroughly.
StoreanyunusedreconstitutedDetectionReagentat4°Cinthedesiccatedpouchandusewithin2months.standardPreparationLabelpolypropylenetesttubesas#1through#8.
Pipet900μLofAssayBufferintotube#1and500μLintotubes#2-#8.
Carefullyadd100μLofthestandardstocksolutiontotube#1andvortexcompletely.
Add500μLoftube#1totube#2andvortexcompletely.
Repeattheseserialdilutionsfortubes#3through#8.
TheconcentrationofN-Acetylcysteineintubes1through8willbe10,000,5,000,2,500,1,250,625,312.5,156.25and78.125nM.useallstandardswithin2hoursofpreparation.std1std2std3std4std5std6std7std8AssayBufferVolume(μl)900500500500500500500500AdditionStockStd1Std2Std3Std4Std5Std6Std7VolumeofAddition(μl)100500500500500500500500FinalConc.(nM)10,0005,0002,5001,250625312.5156.2578.125®8EXPECTASSAYARTISTRY

AssayProcedure

Werecommendthatallstandardsandsamplesberuninduplicatetoallowtheendusertoaccuratelydeterminethiolconcentrationsaccurately.
1.Aplatelayoutsheethasbeenincludedintheinsertonthebackpageoftheinserttoaidpropersampleandstandardidentification.Setplateparametersfora96-wellCorningCostar3686plate.Seewww.arborassays.com/resources/#general-infoforplatedimensiondata.
2.Pipet100μLofsamples,AssayBufferastheblankorstandardsintowellsintheblackplate.
3.Add25μLoftheThioStarReagenttoeachwellusingarepeaterormultichannelpipet.
4.Gentlytapthesidesoftheplatetoensureadequatemixingofthereagents.
5.Covertheplatewiththeplatesealerandincubateatroomtemperaturefor30 minutesinthedark.
6.Setplateparametersfora96-wellCorningCostar3686plate.Seewww.arborassays.com/resources/#general-infoforplatedimensiondata.Readthefluorescentsignalfromeachwellinaplatereadercapableofreadingthefluorescentemissionat510nmwithexcitationat370-410nm.Pleasecontactyourplatereadermanufacturerforsuitablefiltersets.
7.Usetheplatereadersbuilt-in4PLCsoftwarecapABIlitiestocalculatethiolconcentrationsforeachsample.

CalculationofResults

AveragetheduplicateFLUreadingsforeachstandardandsample.
Createastandardcurvebyreducingthedatausingthe4PLCfittingroutineontheplatereader,aftersubtractingthemeanFLUsforthezerostandard.
Thesampleconcentrationsobtainedshouldbemultipliedbythedilutionfactortoobtainneatsamplevalues.

RestrictionsForResearchUseonly
PrecautionofUseAswithallsuchproducts,thiskitshouldonlybeusedbyqualifiedpersonnelwhohavehadlaboratorysafetyinstruction.
Thecompleteinsertshouldbereadandunderstoodbeforeattemptingtousetheproduct.
Dimethylsulfoxideisapowerfulaproticorganicsolventthathasbeenshowntoenhancetherateofskinabsorptionofskin-permeablesubstances.
Wearprotectivegloveswhenusingthesolventespeciallywhenitcontainsdissolvedchemicals.thiostar®thiolDetectionreagentshouldbestoredat4°Cinthedesiccatedpouch.
Allowdesiccatedpouchtowarmtoroomtemperaturepriortoopening.thiostarwillreactwithstrongnucleophiles.
Bufferscontainingthepreservativessodiumazide,Proclin™andKathon™willreactwiththesubstrate.
ReconstitutedThioStarinDMSOstoredat4°Cinthesupplieddesiccatedpouchcanbeusedupto2monthslater.
ThebackgroundonthereconstitutedThioStarwillincreaseslowlyovertimebuttheincreasewillnotaffecttheassayresultsobtained.
Storage4°C
StorageCommentAllcomponentsofthiskitshouldbestoredat4°Cuntiltheexpirationdateofthekit.DMSOwhenstoredat4°Cwillfreeze.Canbestoredtightlycappedatroomtemperature.
SupplierImages
image for Thiol Fluorescent Detection Kit (ABIN577645)ThiolFluorescentDetectionKit
Productcitedin:Liberman,Hamad-Schifferli,Thorsen,Wick,Carr:"InsitumicrofluidicSERSassayformonitoringenzymaticbreakdownoforganophosphates."in:Nanoscale,Vol.7,Issue25,pp.11013-23,2015(PubMed).

Ferraresi,Parizotto,PiresdeSousa,Kaippert,Huang,Koiso,Bagnato,Hamblin:"Light-emittingdiodetherapyinexercise-trainedmiceincreasesmuscleperformance,cytochromecoxidaseactivity,ATPandcellproliferation."in:Journalofbiophotonics,Vol.8,Issue9,pp.740-54,2015(PubMed).

El-Seweidy,Sadik,Shaker:"Roleofsulfurousmineralwaterandsodiumhydrosulfideaspotentinhibitorsoffibrosisintheheartofdiabeticrats."in:Archivesofbiochemistryandbiophysics,Vol.506,Issue1,pp.48-57,2011(PubMed).


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