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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.
Horseradishperoxidase(HRP)conjugatesarepreparedbyamodifiedNakaneandKawaoiprocedure(J.Histochem.Cytochem.1974.22,1084).Peroxidaseconjugatesarecommonlyusedforimmunohistochemistry,Westernblotting,andELISA.Affinity-purifiedanti-horseradishperoxidaseandconjugatesareavailablefordetectionofhorseradishperoxidaseantigenorforsignalamplificationofHRP-containingreagents.Forimmunostainingofmammaliancells,anadvantageofusinganti-horseradishperoxidaseisreducedbackground,sincetheantibodydoesnotrecognizetheendogenousperoxidase-likeenzymesfoundinthosecells.