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1、MethodsinMolecularBiologyMethodsinMolecularBiologyTMVOLUME138ChemokineChemokineProtocolsProtocolsEditedbyAmandaE.I.ProudfootTimothyN.C.WellsimothyN.C.WellsChristineA.PowerHUMANAPRESSPurificationofChemokines11PurificationofChemokinesfromNaturalSources
2、Jens-M.Schr?der1.IntroductionChemokines(e.g.,IL-8)wereoriginallyidentifiedaschemotacticproteinsobtainedfromdifferentnaturalsources.ThesewerepurifiedbytheuseofabioassayfollowedbyN-terminalsequencingandthenmolecularcloninggiv-ingthefullsequenceandmakin
3、gitpossibletoexpressthechemokineasarecombinantprotein.Inthiswayweweresurethatthebiologicallyactivechemokineisreallyreleasedfromnaturalsources.Today,usingthegenomewalkingstrategy,wehavediscoveredanever-increasingnumberofnovelgenesencodingchemokinestha
4、twereexpressedinbacteriaoreukaryoticcellsandsubsequentlytestedastherecombinantformforitsbiologicalactivity.Followingreversetranscriptase-polymerasechainreaction(RT-PCR)inves-tigations,Northernblotexperiments,andinsituhybridizationexperimentsmuchknowl
5、edgeisobtainedaboutlocalizationofchemokinegeneexpression.Antibodies(oftenraisedagainstchemokinepeptidefragmentsorrecombinantmaterial)provideinformationabouttissuelocalizationofimmunoreactivechemokinesbyimmunostainingandpresumethereleaseofimmuno-react
6、ivechemokineprotein.Usuallybiologicalsignificanceoftheconsideredchemokineisextrapolatedfromthesedata.Inprincipal,however,allthesedataareindirect.Thus,itisveryimportanttoprovidedirectproofofthereleaseofbiologicallyactivechemokinesfromnaturalsources.Pu
7、rificationofchemokinesfromnaturalsourcesrequiresspecialstrategies.Firstofall,thedetectionsystemshouldbeusefultodetecttherequiredchemokineatlowconcentrations,i.e.,itisimportanttochooseanassaysystemthatallowsthedetectionoflowamountsofthechemokineinascr
8、eeningsys-tem.ScreeningsystemsoftenusedareBoydenchamberchemotaxisassayFrom:MethodsinMolecularBiology,vol.138:ChemokineProtocolsEditedby:A.E.I.Proudfoot,T.N.C.Wells,andC.A.Power?HumanaPressInc.,Totowa,NJ12Schr?dersystems(seeChapter11and[1]).Beforestar
