| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 680/None |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | R/D/L |
| Category | ProteinBiochemistry Generalproteins |
| Related |
Uponreceipt,storeThiolBlue™(ComponentA)at-20°C,keepfromlightandmoisture.Whenstoredproperly,thekitcomponentsshouldbestableforsixmonths.
Note1:DonotfreezeSpinColumn(ComponentC).
Note2:Warmallthecomponentsbeforeruntherequiredassays.50to100gproteinsampleisneededfordeterminingtheamountofthiolamount.
1.PrepareSampleSolution:
1.1Use50to100μgproteinsample.
1.2Adjustthevolumeto100μLwithAssayBuffer(ComponentB).
Note:TheproteinsampleshouldbeinpH=6.0bufferandwithoutDTTorotherreagentcontainingfreethiols. 2.RunThiolAssay: 2.1Addtheproteinsample(fromStep1.2)toonevialofThiolBlue™(ComponentA). 2.2Mixthemwellbyrepeatedlypipettingforafewtimesorvortexthevialforafewseconds. 2.3Keepthereactionmixtureatroomtemperatureandrotateorshakefor30-60minutes. 3.PrepareSpinColumnforSamplePurification: 3.1InverttheSpinColumn(ComponentC)severaltimestoresUSPendthesettledgelandremoveanybubbles. 3.2SnapoffthetipandplacethecolumnintheWashingTube(2mL,ComponentD).Removethecaptoallowtheexcesspackingbuffertodrainbygravitytothetopofthegelbed.Ifcolumndoesnotbegintoflow,pushcapbackintocolumnandremoveitagaintostarttheflow.Discardthedrainedbuffer,andthenplacethecolumnbackintotheWashingTube.However,centrifugeimmediatelyifthecolumnisplacedintoa12x75mmtesttube(notprovided). 3.3Centrifugefor1mininaswingingbucketcentrifugeat1,000xg(seeCentrifugationNotessection)toremovethepackingbuffer.Discardthebuffer. 3.4Apply1mLAssayBuffer(ComponentB)tothecolumn,letthebufferdrainoutbygravity,orcentrifugethecolumnfor1mintoremovethebuffer.Discardthebufferfromthecollectiontube.Repeatthisprocessfor3-4times. 3.5Centrifugefor2minutesinaswingingbucketcentrifugeat1,000xg(seeCentrifugationNotessection)toremovethereactionbuffer.Discardthebuffer. Note1:SpinColumn(ComponentC)canfitinto2mLmicrocentrifugetubesor12x75mmtesttubesforsamplecollectionduringcentrifugation.Usethe2mLmicrotubesprovidedwiththecolumnsfortheinitialcolumnequilibrationstep. Note2.Swingingbucketcentrifugescapableofgeneratingaminimumforceof1,000xgaresuitableforBio-Spincolumnuse.Thegravitationalforcecreatedataparticularrevolutionspeedisafunctionoftheradiusofthemicrocentrifugerotor.Consulttheswingingbucketcentrifugeinstructionmanualfortheinformationaboutconversionfromrevolutionsperminute(RPM)tocentrifugalorg-force.Alternatively,usetheequation(1)tocalculatethespeedinRPMrequiredtoreachthegravitationalforceof1,000xg. RCF(g)=(1.12×〖10〗^(-5)〖)×(RPM)〗^2×r(1) RCF=therelativecentrifugalforce RPM=thespeedoftherotor r=theradiusincentimetersmeasuredfromthecenteroftherotortothemiddleoftheBio-Spincolumn 4.PurifyReactionProduct: 4.1Placethecolumn(fromStep3.5)inacleanCollectingTube(1.5mL,ComponentE).Carefullyloadthesample(100μL)directlytothecenterofthecolumn. 4.2Afterloadingthesample,add10μLAssayBuffer(ComponentB)tothetopandcentrifugethecolumnfor5minat1,000xg,andcollectthesolutionintothecollectingtube. 5.RunAbsorptionSpectrawith0.2mLor0.5mLQuartzCuvette: 5.1Dilutethereactionproduct(fromStep4.2)by5-foldswithAssayBuffer(ComponentB)dependingonthecuvettesizeusedandtheabsorbancereading. Note:Thedilutionfactordoesn’taffectthefinalthiolquantitationresult. 5.2Measuretheabsorptionspectrumfrom250to750nm,oronlyreadtheabsorbancenumberat280nmand680nm. ActivationoftranscriptionfactorsinhumanbronchialepithelialcellsexposedtoaqueousextractsofmainstreamcigarettesmokeinvitroReferences&Citations 
Authors:TakashiSekine,TadashiHirata,ToshikiMine,YasuoFukano
Journal:Toxicologymechanismsandmethods(2016):22--31
