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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.
Cy5conjugatesareexcitedmaximallyat650nmandfluorescemaximallyat670nm.Theycanbeexcitedtoabout98%ofmaximumwithakrypton/argonlaser(647nmline)ortoabout63%ofmaximumwithahelium/neonlaser(633nmline).Cy5canbeusedwithavarietyofotherfluorophoresformultiplelabelingduetoawideseparationofitsemissionfromthatofshorterwavelength-emittingfluorophores.However,werecommendAlexaFluor®647asthepreferredfar-red-fluorescingdyebecauseitisbrighterthanCy5inaqueousmountingmedia.Thereverseistruewhenmountinginnon-polar,plasticmountingmedia,i.e.Cy5isbrighterthanotherfar-red-fluorescingdyes,includingAlexaFluor®647andDyLight650.
