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WholeIgGantibodiesareisolatedasintactmoleculesfromantiserabyimmunoaffinitychromatography.TheyhaveanFcportionandtwoantigenbindingFabportionsjoinedtogetherbydisulfidebondsandthereforetheyaredivalent.Theaveragemolecularweightisreportedtobeabout160kDa.ThewholeIgGformofantibodiesissuitableforthemajorityofimmunodetectionproceduresandisthemostcosteffective.
Storefreeze-driedpowderat2-8°C.Whenreadytouse,rehydratewithindicatedvolumeofd.waterandcentrifugeifnotclear.Productisstableforabout6weeksat2-8°Casanundilutedliquid.Prepareworkingdilutionfresheachday.Forextendedstorageafterrehydration,addanequalvolumeofglycerol(ACSgradeorbetter)forafinalconcentrationof50%,andstoreat-20°Casaliquid.Note:aftertheadditionofglycerol,theconcentrationofproteinandbuffersaltsisone-halfoftheoriginal.Alternatively,aliquotandfreezetheproductat-70°Corbelowintheabsenceofglycerol.Avoidrepeatedfreezingandthawing.
Expirationdate:oneyearfromdateofrehydration.However,theexpirationdatemaybeextendediftheproductisstoredaccordingtotherecommendationandthetestresultsareacceptableforitsintendeduse.
RRX(RhodamineRed-X)conjugateshaveapeakofexcitationat570nmandapeakofemissionat590nm.AlthoughTRITChasbeenusedtraditionallywithFITCfordoublelabeling,bettercolorseparationisachievedbyusingRRXorAlexaFluor®594.RhodamineRed-Xisparticularlyusefulfor3-and4-colorlabelingwithDyLight405,AlexaFluor®488,andAlexaFluor®647byusingaconfocalmicroscopeequippedwitha405nmlaserandakrypton/argonlaser.FluorescencefromRRXliesaboutmidwaybetweenthatofAlexaFluor®488andAlexaFluor®647,anditshowslittleoverlapwitheitherdye.Thekrypton-argonlaseremitslinesat488nm,568nm,and647nm,whichareoptimalforexcitingAlexaFluor®488,RRX,andAlexaFluor®647,respectively.Byaddinga405nmlaseranda420nmemissionfilter,4-colorlabelingispossibleusingDyLight405-conjugatedsecondaryantibodiesfromJIR(Figure5).Theseparationbetweenallfourdyesisperfectfor4-colorlabeling,andallfourdyesareverybright.
