SampleGelInformation:

Overview:

Lactadherinisawidelydistributedglycoprotein(~50kDa),whichwasoriginallycharacterizedduetoitsassociationwithmilkfat/lipidglobulemembranes.SynonymousnamesarePAS-6/7,bovine-associatedmucoprotein,BA-46,P47,andMFG-E8.Structuralhallmarksoflactadherinarethepresenceoftwoepidermalgrowthfactor(EGF)homologydomains(withanRGDpeptidemotifinthesecondEGFdomain),andtwoCdomainssharinghomologywiththediscoidinfamilyoflectindomainsincludingthephospholipid-bindingdomainsofbloodclottingfactorsVandVIII.Lactadherinshowspreferentialbindingtophosphatidylserine(L-form)inacalciumindependentmanner,andbindsmorespecificallythanAnnexinV.

Purifiedlactadherinfunctionsasananticoagulantbyblockingphosphatidylserine-containingmembranesitesforbloodcoagulationproteins(10).Fluoresence-labeledlactadherinfunctionsasasensitiveprobeforexposedphosphatidylserineonnucleatedcellsandonstimulatedplatelets(8,9).LactadherinwillbindtomembranesthathavephosphatidylserinecontentbelowthethresholdforannexinVbinding.

Lactadherinispurifiedfromun-pasteurizedbovinemilk(11).

IllustratedApplications

Above:K562cells(top)andHL60cells(bottom)co-stainedwithbothFITC-conjugatedlactadherin(green)andAlexa-647conjugatedannexinV(red)earlyinapoptosis.Theannexinisinternalizedingranulesandisnotdetectablystainingthecells. Reference:Shi,J.,Y.Shi,L.N.Waehrens,J.T.Rasmussen,C.W.HeegaardandG.E.Gilbert(2006)."Lactadherindetectsearlyphosphatidylserineexposureonimmortalizedleukemiacellsundergoingprogrammedcelldeath."CytometryA69(12):1193-201. Copyright2006. JohnWiley&Sons,Inc. ReprintedwithpermissionofJohnWiley&Sons,Inc.

Above:HeLacellsstainedwithFITC-conjugatedlactadherin2hours(top)and3hours(bottom)aftertreatmentwithstaurosporine.Earlyinapoptosisthecellshavesmallvesiclesandlong,thinappendagesthatstainavidlyforlactadherin.Reference:WaehrensLN,Heeghaard,CW,GilbertGE,RasmussenJT.BovineLactadherinasaCalcium-independentImagingAgentofPhosphatidylserineExpressedontheSurfaceofApoptoticHeLaCells2009J.Histochem.Cytochem.(ePubJune2009).

Above:Phosphatidylserineexposureinmousemesentericvenousthrombosis. Miceweregiven1µgeachoflactadherinandannexinVbytailveinimmediatelypriortoexternalizationofthemesentery.Themesentarywasexposedtoferricchlorideandthentheanimalswereperfusedwithsaline/paraformaldehyde.Serialsectionsofthemesentarywerestainedwithanti-fibrinogen/fibrin(left),anti-platelet(middle),andanti-lactadherinantibodies(right)developedwiththealkalinephosphataseVectorRedsubstrate.Alayeroffibrinogen/fibrin(left,closedarrows)overlaidamuralhemorrhage(openstar).Platelets(middle)werescatteredalongtheluminalsurfaceofthethrombus(opentriangles)aswellasuponfibrinogen/fibrinstrandsextendingintothelumen.Lactadherinstaining(right)wasstrongestalongtheraisedendotheliumsurface(closedarrow),includingadherentplateletsclosetothewall.Plateletsonfibrinstrandsdidnotstaindetectably(openarrow).(ShiJ,PipeSW,RasmussenJT,HeegaardCW,GilbertGE.Lactadherinblocksthrombosisandhemostasisinvivo:correlationwithplateletphosphatidylserineexposure.JThrombHaemost.Jul2008;6(7):1167-1174).

Properties:

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