JournalofVisualizedExperimentswww.jove.comCopyright©2013CreativeCommonsAttribution-NonCommercialLicenseFebruary2013|72|e50031|Page1of6VideoArticleDetectionofProteinPalmitoylationinCulturedHippocampalNeuronsbyImmunoprecipitationandAcyl-BiotinExchange(ABE)G.StefanoBrigidi1,ShernazXBamji11DepartmentofCellularandPhysiologicalSciences,BrainResearchCentre,UniversityofBritishColumbiaCorrespondenceto:ShernazXBamjiatshernaz.bamji@ubc.caURL:http://www.jove.com/video/50031DOI:doi:10.3791/50031Keywords:Neuroscience,Issue72,Biochemistry,Neurobiology,MolecularBiology,CellularBiology,Physiology,Proteins,synapse,culturedhippocampalneurons,palmitoylation,lipid,immunoprecipitation,westernblotting,biotin,Acyl-BiotinExchange,ABE,neuron,brain,cellculture,rat,mouse,animalmodelDatePublished:2/18/2013Citation:Brigidi,G.S.,Bamji,S.X.DetectionofProteinPalmitoylationinCulturedHippocampalNeuronsbyImmunoprecipitationandAcyl-BiotinExchange(ABE).J.Vis.Exp.(72),e50031,doi:10.3791/50031(2013).AbstractPalmitoylationisapost-translationallipidmodificationinvolvingtheattachmentofa16-carbonsaturatedfattyacid,palmitate,tocysteineresiduesofsubstrateproteinsthroughalabilethioesterbond[reviewedin1].Palmitoylationofasubstrateproteinincreasesitshydrophobicity,andtypicallyfacilitatesitstraffickingtowardcellularmembranes.Recentstudieshaveshownpalmitoylationtobeoneofthemostcommonlipidmodificationsinneurons1,2,suggestingthatpalmitateturnoverisanimportantmechanismbywhichthesecellsregulatethetargetingandtraffickingofproteins.Theidentificationanddetectionofpalmitoylatedsubstratescanthereforebetterourunderstandingofproteintraffickinginneurons.Detectionofproteinpalmitoylationinthepasthasbeentechnicallyhinderedduetothelackofaconsensussequenceamongsubstrateproteins,andtherelianceonmetaboliclabelingofpalmitoyl-proteinswith3H-palmitate,atime-consumingbiochemicalassaywithlowsensitivity.DevelopmentoftheAcyl-BiotinExchange(ABE)assayenablesmorerapidandhighsensitivitydetectionofpalmitoylatedproteins2-4,andisoptimalformeasu...
