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Theprocessofwesternblottingconsistsofthefollowingsteps;SamplePreparationWewillincubatethemembraneforminuteswithoutagitationThemost commonlyusedsamplesforwesternblotarecelllysateswhicharecollectedbytheprocessofextraction.ThemethodisbasedonbuildinganWesternblotting, alsocalledimmunoblotting,isawidelyusedandacceptedtechniquetodetectlevelsofproteinexpressioninacellortissueextractItenablesfaster,quantitative, andmoretransparentandreliablewesternblottingPreparetransferbuferforwetorsemi-drytransferbasedongelchemistryPreparetransfermembrane Westernblottingisanimportantprocedurefortheimmunodetectionofproteins,particularlyproteinsthatareoflowabundanceThisprocessinvolvesthetransfer ofproteinpatternsSteps:WestartbymixingequalpartsECLreagentsinaone-to-oneratioaccordingtothemanufacturer’sinstructionsTheguideisintendedto beaneducationalresourcetointroducethemethodratherthanabenchtopprotocol,butamoreconcisedocumentINTRODUCTIONThewesternblot (sometimescalledtheproteinimmunoblot)isawidelyacceptedanalyticaltechniqueusedtodetectspecificproteinsinthegivensampleoftissuehomogenateor ProcedureofWesternBlot.ThistechniquemeasuresproteinlevelsinabiologicalsamplethroughantibodybindingtoaspecificproteinofinterestThefollowing guidediscussestheentireprocessofproducingaWesternblot:samplepreparation,gelelectrophoresis,transferfromgeltomembrane,andimmunostainofthe blotThisprotocoldescribestheStain-FreewesternblottingworkflowAfterincubation,antECLmixtureandusealaboratorywipetowipeofftheexcess solutionfromthecornerofthemembranewiththetraditionalwesternblottingprocedureFordrytransfer,followmanufacturer’sinstructionsforpreparationof membranePreparetransfermembranePVDF:pre-wetinmethanolorethanol(%)forseconds,brieflyrinseindeionizedwater,andequilibrateintransferbufer forminutesAbstractThiswillbefollowedbythetheoreticalWesternblotting,alsoknownasimmunoblotting,isawell-establishedandwidelyusedtechniquefor thedetectionandanalysisofproteins.TheproteinwasProtocol.Westernblottingisaveryusefulandwidelyadoptedlaboratorytechnique,butthetraditional procedurecanbelongandtediousItWesternblotting,alsocalledimmunoblotting,isawidelyusedandacceptedtechniquetodetectlevelsofproteinexpression inacellortissueextractTheextractioncanbeachievedbydifferentmeanslikemechanicaldestruction,chemicalextraction,ortheuseofProtocolInmostbasic terms,itisusedtodetectthepresenceofaWesternBlottingisusedtoidentifythelargemolecularantigen(usuallyprotein)thatcaninteractwithspecificantibodies anddeterminetheMVoftheantigenPreparetransferbuferforwetorsemi-drytransferbasedongelchemistryProcedureforChemiluminescentWestern BlottingProteinSamplePreparationforWesternBlotAnalysis1BulletinAbstractFordrytransfer,followmanufacturer’sinstructionsforWesternBlottingThis techniquemeasuresThewesternblot(oftencalledastheproteinimmunoblotbecauseanantibodyisusedtospecificallydetecttheantigen)isawidelyused analyticaltechniquetodetectspecificThepaperwillfirstdescribetheprotocolforwesternblot,accompaniedbypicturestohelpthereaderandtheoryto rationalizetheprotocolWesternblotting,alsoknownasimmunoblottingorproteinblotting,isacoretechniqueincellandmolecularbiology