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1、METHODSINMOLECULARBIOLOGYMETHODSINMOLECULARBIOLOGYTMTMVolume264ProteinProteinArraysArraysMethodsandProtocolsMethodsandProtocolsEditedbyEditedbyEricT.Fung,EricT.Fung,MDMD,,PPhhDDProteinArraysFromcDNAExpressionLibraries11ProteinArraysFromcDNAExpressionLibrariesHendrikWeiner,Thom
2、asFaupel,andKonradBüssowSummaryThischapterdescribestheproductionofacDNAexpressionlibraryfromhumanfetalbrain,theconstructionofahigh-densityproteinarrayfromsuchalibrary,andtwoapplicationstoscreenthearrayforbindingproteins.Afterproducingthelibraryanddecollatingtheexpressionclones
3、,onecanpickthousandsofexpressioncloneswithalaboratoryrobotandcandepositthemintomicrotiterplatesinanorderedmanner.Suchorderedclonelibrariesarethestartingmaterialfortheconstructionofahigh-densityproteinarray.Thisarrayisconstructedbyspot-tingtheexpressionclonesontoaprotein-bindin
4、gmembrane.Followingcellgrowthandinduc-tionofproteinexpressiononthemembrane,thecellspotsarelysedandtheirrecombinantproteinimmobilizedonthemembrane.Theso-constructedarraycarriesthousandsofproteinswithouttheneedtoclone,express,andspotindividualproteins.Sucharraysallowonetoscreenf
5、ornumerousproteinfunctionsinahigh-throughputmanner.KeyWords:Proteinarray;cDNAexpressionlibrary;high-densityspotting;clonearray;proteinantigen;proteinfunction;protein–proteininteraction;posttranslationalmodification;high-throughputscreening.1.IntroductionArraysofcomplementaryDN
6、A(cDNA)expressionlibrariescarrythousandsofproteinswithouttheneedtoclone,express,andspotindividualproteins(1).Thesearraysarepracticalformatstoscreenenmasseforagivenproteinfunction,thatis,toidentifyproteinantigens(1,2),includingautoantigens(3),bindingproteins(4),andsubstratesfor
7、argininemethyltransferases(5).Althoughnotyetdemonstrated,thearraysmayalsopermitstudiesonposttranslationalmodificationsotherthanproteinmethylation,thatis,tofindsubstratesforcertainproteinkinases.TheproteinarraysdescribedherearemadeusingcDNAlibrariesthatarecon-structedinexpressi
8、onvectors.Withthehelpofalaboratoryrobot,onecanpickthou-sandso
