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1、MethodsinMolecularBiologyMethodsinMolecularBiologyTMVOLUME171ProteoglycanProteoglycanProtocolsProtocolsEditedbyEditedbyRenatoV.Iozzo,RenatoV.Iozzo,MDMDHUMANAPRESSHUMANAPRESSIsolationfromCellCulturesandTissues31IsolationofProteoglycansfromCellCulturesandTissuesMa
2、sakiYanagishita1.IntroductionProteoglycansareaclassofglycosylatedproteinscharacterizedbythepresenceofglycosaminoglycansasacarbohydratecomponent,whichendowthemwithuniquebiologicalaswellasbiochemicalproperties.Therefore,isolationofproteoglycansfromvariousbiologica
3、lsourcessuchascellculturesandtissuescouldbeachievedbyordinarymolecularpurificationproceduresutilizingtheirgeneralmolecularproper-tiesandbythosetakingadvantagesofthepresenceofglycosaminoglycanmoiety.Thischapterfocusesonthelatterexperimentalprocedures,whicharepart
4、icularlyuse-fulforobtainingtotalproteoglycanandglycosaminoglycanspeciesfromvariousbiologicalsources.Theseprotocolscouldbefollowedbypurificationproceduresspecifictoindividualproteoglycanspecies(i.e.,byusingantibodiestocoreproteins,orbindingproteinstospecificseque
5、ncesofglycosaminoglycans)toselectspecificmolecules.Twomajorclassesofwell-establishedpurificationproceduresaimingatthepres-enceofglycosaminoglycansinthemoleculehavebeenusedextensively.Theyinclude(1)densitygradientultracentrifugation,and(2)anion-exchangechroma-tog
6、raphy.Theformerproceduremakesuseofthefactthattheglycosaminoglycanmoietyofproteoglycanshashighspecificgravity,so,proteoglycanswithalargenumberofglycosaminoglycanshavehighmoleculardensities(oftenashighasthoseofnucleicacids).Therefore,suchaprocedureisparticularlysu
7、itedforthepurificationofproteoglycanswithmanyglycosaminoglycanchains(e.g.,aggrecan,themajorproteoglycanincartilagetissues).Ontheotherhand,theprocedureisnotsuitableforisolatingproteoglycanswithonlyfewglycosaminoglycansandhighproteincontents(e.g.,“small,leucine-ri
8、chproteoglycans”andcellsurfaceheparansulfateproteoglycans).Purificationproceduresbelongingtothelatterclasstakeadvantageofhighnegativechargescontributedbysulfategroups
