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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.
AlexaFluor®647-conjugatedantibodiesabsorblightmaximallyaround651nmandfluorescemaximallyaround667nm.TheyarebrighterthanCy5andDyLight650inaqueousmountingmedia.AlexaFluor®647-andAPC-conjugatedsecondaryantibodiesarethebestchoiceforflowcytometrywhensecondaryantibodiesfluorescingatthesewavelengthsaredesired.AlexaFluor®647conjugatesarethebestchoiceoffarred-emittingdyesformultiple-labelingdetectionwithaconfocalmicroscope.
AsignificantadvantageofusingAlexaFluor®647overlowerwavelength-emittingdyesisthelowautofluorescenceofbiologicalspecimensinthisregionofthespectrum.However,becauseofitspeakemissionat667nm,AlexaFluor®647cannotbeseenwellbyeye,anditcannotbeexcitedoptimallywithamercurylamp.Therefore,AlexaFluor®647isnotrecommendedforusewithconventionalepifluorescentmicroscopes.Itismostcommonlyvisualizedwithaconfocalmicroscopeequippedwithanappropriatelaserforexcitationandafar-reddetector.AlexaFluor®647conjugatesarelessexpensivealternativestoallophycocyaninconjugatesforflowcytometry.
