DescriptionTheFollowingProtocolcanbeusedtocoupleeitherFITCorBiotintoIgG.Thesamemethodisusedforboth,andwhendiscrepanciesoccur,theywillbenoted.Procedure1.Dissolve2mgofIgGin1mLofPBS(pH8.0)inclean16x125mmpolypropylenetesttube.Ifneeded,adjustpHto8.0using0.5Mcarbonatebuffer.2.Preparefresh,1mg/mLofeitherNHS-FITCinDMSOor1mg/mLNHS-LC-BiotininDH2O.IfFITCcoupling,add75uLofFITC/DMSOsolutiontoAntibodywhilevortexing.IfBiotinylating,add150uLofBiotin/WatersolutiontoAntibodywhilevortexing.3.Incubateatroomtemperaturefor45minutes.4.ThenextstepistoseparatethecoupledfromuncoupledFITCorBiotin.Thiscanbedonein2ways.ThefirstistoDialyzethecoupledantibodyagainstPBSfor24hourswith3changesofPBS.Doingthiswillnotchangeconcentrationoforiginalantibody(recommendedforantibodywithaconcentrationlessthan1mg/mL).ThesecondwayistouseaPD-10column.Usingthismethodwilldilutetheantibodybyafactorof2.UsingaPD-10column:a)WashPD-10columnwith30mLofPBS.b)Addthe1mLFITC-IgorBiotinIgsolutionafterthetopofcolumnisalmostdry.ClosestopcocktostopflowaftertheAntibodyhaselutedintothecolumn.c)Add5mLofPBSontop.d)OpenStopcock.collectin2mLfractions,discardingthefirst2mLandkeepingthe2nd2mLfraction.Thesecond2mLfractioncontainstheconjugatedantibody*.*NOTE:Stepd)appropriateonlyif1mLofconjugated-Igisaddedtothecolumn.If2mLofconjugated-Igisadded,discardfirst1mLandkeepthenext4mL.Theantibodyisalwaysdilutedby2xafterleavingthecolumn.5.WhenFITCcouplinganantibody,youcandeterminetheFITCtoProteinratio.WhenBiotinylatinganantibody,youcantesttheantibodybyFlowCytometry.6.F/PRatio:a)DiluteanaliquotofFITC-IgcomplexwithbuffersothattheAbsorbanceat280nm(A280)islessthan2.0b)DetermineandrecordA280andA492.c)Calculateproteinconcentration:protein(mg/mL)=(A280-(A492x0.35))/1.4d)Calculatemolesofprotein:=protein(mg/mL)/1.5x10^5e)CalculatemolesofFITC:=A492/0.69x10^5f)DetermineF/Pratio:=molesofFITC/molesofproteing)F/Pratioshouldbebetween5:1and6:1forFlowCytometricuse.

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