资源描述：
《MMB 138 Chemokine Protocols》由会员上传分享，免费在线阅读，更多相关内容在学术论文-天天文库。
1、MethodsinMolecularBiologyMethodsinMolecularBiologyTMVOLUME138ChemokineChemokineProtocolsProtocolsEditedbyAmandaE.I.ProudfootTimothyN.C.WellsimothyN.C.WellsChristineA.PowerHUMANAPRESSPurificationofChemokines11PurificationofChemokinesfromNaturalSources
2、Jens-M.Schröder1.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,NJ12Schrödersystems(seeChapter11and[1]).Beforestar
