fortebio-生物素化蛋白及SA传感器应用

Assayontheinteractionofbiotin-proteinandothermolecularOverviewMaterialsEZ-LinkNHS-PEG4-Biotin(Thermo,partno.21329)preparedasa1mMsolutioninwaterCanalsousetheNHS-PEG12-Biotin(Thermo,partno.21312)orSulfo-NHS-LC-LC-Biotin(Thermo,partno.21338)orNHS-LC-LC-Biotin(Thermo,partno.21343)Distilledwater1XPBSDMSOZebadesaltingspincolumnsforbufferexchangeForproteinsamplesof30–130μL,use0.5mLcolumns(Thermo,partno.89882)Forproteinsamplesof200–700μL,usethe2mLcolumns(Thermo,partno.89889)Forproteinsamplesof600–2000μL,usethe5mLcolumns(Thermo,partno.89891)Ligand:proteinAnalyte:othermolecular1.5mLmicrocentrifugecollectiontubesStreptavidinSensor(Pall,partno.18-501918-502018-5021)KineticBuffer(1XPBS+0.02%Tween20+0.1%BSA)RegenerationBuffer(10mMGlypH1.7-2.5)(Optional)Slide-A-Lyzer(Thermo,partno.66370)ProcedureBiotinylationofProteinforImmobilizationontoStreptavidinBiosensorsProteinSamplePreparationEnsurethatproteintobebiotinylatedrequiresatleast50ugtotalataconcentrationofatleast100ug/mL,andthemolecularofitis=7KDaEnsurethattheproteiniscarrier-proteinfree(forantibodiescontainingcarrierprotein,seeForteBioTechnicalNote11.)Ensurethattheproteintobebiotinylatedisnotinabuffercontainingprimaryamines(i.e.,Trisorglycine).Ifproteinisinabuffercontainingprimaryamines,exchangeintoPBSeitherbydialysisordesaltingspincolumns.Theproteinconcentrationisrecommendedtobeatleast1mg/mLforthisprocess.ProteinSamplePreparationbyDialysis(recommendedforsamples0.5mL)Dialyzeeachsample1:1000in1XPBSusingaSlide-A-Lyzer.AllowtheproteinandSlide-A-LyzertostirgentlyinPBSforaminimumof3hoursbeforechangingthePBS.ChangePBSatleast4timesbeforeextractingtheproteinProteinSamplePreparationbyDesaltingColumn(recommendedforsamples0.5mL)DesaltingorBufferExchangeProcedureA.SpinColumnPreparationRemovecolumn’sbottomclosureandloosencap(donotremovecap).Placecolumnina1.5-2.0mLcollectiontube.Centrifugeat1500×gfor1minutetoremovestoragesolution.Blotbottomofcolumntoremoveexcessliquid.Placeamarkonthesideofthecolumnwherethecompactedresinisslantedupward.Placecolumninthemicrocentrifugewiththemarkfacingoutwardinallsubsequentcentrifugationsteps.Improperorientationwillresultinreduceddesaltingefficiency.ThefollowingstepsareforbufferexchangeAdd300μLofbufferontopoftheresinbed.Centrifugeat1500×gfor1minutetoremovebuffer.RepeatStep4twotothreeadditionaltimes,discardingbufferfromthecollectiontube.B.SampleLoadingPlacecolumninanewtube,removecapandslowlyapply30-130μLofsampletothecenterofthecompactedresinbed.(Optional)Forsamplevolumes70μLapplya15μLstackerofultrapurewaterorbuffertothetopoftheresinbedafterthesamplehasfullyabsorbedtoensuremaximalproteinrecovery.Centrifugeat1500×gfor2minutestocollectdesaltedsample.Discarddesaltingcolumnafteruse.BiotinCalculationandPreparationTheamountof1mMbiotinreagentrequireddependsonthemolarcouplingratio(MCR)ofthebiotinreagenttoprotein.Ingeneral,itisrecommendedtousea1:1ratio(1biotinforeveryproteinmolecule).Iftheproteinconcentrationislessthan500μg/mLoriftheextentofbiotinylationatanMCRof1:1isfoundtobeinsufficient,a3:1or5:1ratioisrecommended.1.BasedontheselectedMCR,calculatethevolumeof10mMbiotinreagentneeded.AsamplecalculationisshowninFigure.Figure1Equationforcalculatingtherequiredvolumeof1mMbiotinreagent.2.Preparea1mMbiotinreagentsolution.IfusingNHS-PEG4-Biotin,piercethefoiltopandadd170μLofdistilledwatertothetubetocreatea20mMstocksolution.Add50μLofthe20mMbiotinstocksolutionto950μLofdistilledwatertocreatea1mMsolutionofNHS-PEG4-Biotin.FortheNHS-PEG12-Biotin,theproductinsertinstructsonhowtocreatea250mMstocksolution;add4μLofthisstocksolutionto996μLwatertocreatea1mMsolution.Forthesulfo-NHS-LC-LC-Biotin,theproductinsertinstructsonhowtocreatea10mMstocksolution;add100μLofthisstocksolutionto900μLdistilledwatertocreatea1mMsolution.FortheNHS-LC-LC-Biotin,dissolve0.5mgreagentin87.5μLofDMSOfor10mM;add10uLofthisstocksolutionto90μLtocreatea1mMsolution.Thestocksolutionkeepin4℃forXmonths.3.Toeachsample,addtheappropriatevolume(μL)ofBiotinreagentascalculatedinStep1.4.Miximmediately.5.Incubate30minutesatroomtemperatureor2hoursonice.6.Stopthereactionbyremovingtheexcessbiotinreagentusingthedesaltingcolumn(followmanufacturer’sproductinsert).RunningaBindingKineticsAssayAtypicalassayprotocolrunsasfollows:Preparebuffers,regenerationsolution,ligandandanalytesamples.Ligandsample:50-100nMor10-50ug/uL,analytesample:1000,100,10nMforoptimationHydratethebiosensorsforatleast10minutes.Hydrationbuffershouldmatchthebufferusedthroughouttheassayascloselyaspossible.Preparetheassayplate,fillingcolumnsofwellswithbuffers,regenerationsolution,ligandandinteractingproteinsamples.SetuptheassayintheOctetDataAcquisitionsoftware:defineplatelayout,defineassaystepsandassignbiosensors.Equilibrateboththehydratedbiosensorassemblyandtheassayplatefor10minutesontheOctetinstrument.Equilibrationallowsthebiosensorstofullyhydrateandtheassayplatetoreachastabletemperature.Runtheassay.Performdataprocessingandanalysis.AtypicalsampleplatelayoutandassaysummaryisshowninFigure2.Figure2A)Platelayoutdiagramforstandard