| Formulation | TBS,1%BSA,0.02%azide,pH7.4 |
| Storage | 4°C |
| Purity | >95%bySDS-PAGE |
| ActivityDetermination | N/A |
| ShelfLife(properlystored) | 12months |
Lactadherin(MFG-E8)binds,Ca++-independently,tophosphatidyl-L-serineviatheC2domainandbindstheavb3andavb5integrinsviathesecondEGFdomain.Theoneletteraminoacidcodesrefertoidentifiedaminoacidsthatparticipateinbinding.FortheC2domainthecapitallettersrefertowater-exposedhydrophobicresidues.
SampleGelInformation:
| Gel | Novex4-12%Bis-Tris |
|---|---|
| Load | BovineLactadherin,1µgperlane |
| Buffer | MOPS |
| Standard | SeeBluePlus2;Myosin(191kDa),PhosphorylaseB(97kDa),BSA(64kDa),GlutamicDehydrogenase(51kDa),AlcoholDehydrogenase(39kDa),CarbonicAnhydrase(28kDa),MyoglobinRed(19kDa),Lysozyme(14kDa) |
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:
| Names | Lactadherin,MFG-E8,PAS-6/7,SED1,p47 | |||||
|---|---|---|---|---|---|---|
| Localization | Milk(1),alsosecretedbystimulatedmacrophages(2),vascularsmoothmuscle(3),endothelialcells(4) | |||||
| Plasmaconcentration | unknowninnormaladults,measurableinwomenwithmetastaticbreastcancer(5),measurableinnewborncalvesaftermilkmeal(1) | |||||
| Modeofaction | Physiologicfunctionasopsoninorbridgeligand(6,7).Thelectin-likedomainsengagephosphatidyl-L-serine,thesecondEGFdomainengagesavb3and/oravb5integrinleADIngtophagocytosisofapoptoticcells. | |||||
| Molecularweight | 52kDaand47kDa(glycosylationvariants,PAS-6andPAS-7respectively) | |||||
| Extinctioncoefficient |
| |||||
| Structure | singlechainwithtwoEGFdomainsandtwoCdomains.ThemouseisoformcontainsacentralprolinerichregionandthehumanmoleculecontainsasingleEGFdomain | |||||
| Percentcarbohydrate | 6%,13%forglycosylationvariants | |||||
| Post-translationalmodifications | glycosylation |
