Western blot原理.docx

Western blot原理.docx

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Westernblot原理

Westernblot

FromWikipedia,thefreeencyclopedia

WesternblotanalysisofproteinsseparatedbySDS-PAGE.[1]

TheWesternblot（alternatively,proteinimmunoblot）isananalyticaltechniqueusedtodetectspecificproteinsinagivensampleoftissuehomogenateorextract.Itusesgelelectrophoresistoseparatenativeordenaturedproteinsbythelengthofthepolypeptide（denaturingconditions）orbythe3-Dstructureoftheprotein（native/non-denaturingconditions）.Theproteinsarethentransferredtoamembrane（typicallynitrocelluloseorPVDF）,wheretheyareprobed（detected）usingantibodiesspecifictothetargetprotein.[2][3]

Therearenowmanyreagentcompaniesthatspecializeinprovidingantibodies（bothmonoclonalandpolyclonalantibodies）againsttensofthousandsofdifferentproteins.[4]Commercialantibodiescanbeexpensive,althoughtheunboundantibodycanbereusedbetweenexperiments.Thismethodisusedinthefieldsofmolecularbiology,biochemistry,immunogeneticsandothermolecularbiologydisciplines.

Otherrelatedtechniquesincludeusingantibodiestodetectproteinsintissuesandcellsbyimmunostainingandenzyme-linkedimmunosorbentassay（ELISA）.

ThemethodoriginatedfromthelaboratoryofGeorgeStarkatStanford.ThenameWesternblotwasgiventothetechniquebyW.NealBurnette[5]andisaplayonthenameSouthernblot,atechniqueforDNAdetectiondevelopedearlierbyEdwinSouthern.DetectionofRNAistermednorthernblottingandthedetectionofpost-translationalmodificationofproteinistermedeasternblotting.

Contents

∙1StepsinaWesternblot

o1.1Tissuepreparation

o1.2Gelelectrophoresis

o1.3Transfer

o1.4Blocking

o1.5Detection

▪1.5.1Twosteps

▪1.5.2Onestep

o1.6Analysis

▪1.6.1Colorimetricdetection

▪1.6.2Chemiluminescentdetection

▪1.6.3Radioactivedetection

▪1.6.4Fluorescentdetection

o1.7Secondaryprobing

∙22-Dgelelectrophoresis

∙3Medicaldiagnosticapplications

∙4Protocols

∙5References

∙6Seealso

o6.1Relatedlinks

StepsinaWesternblot

Tissuepreparation

Samplesmaybetakenfromwholetissueorfromcellculture.Inmostcases,solidtissuesarefirstbrokendownmechanicallyusingablender（forlargersamplevolumes）,usingahomogenizer（smallervolumes）,orbysonication.Cellsmayalsobebrokenopenbyoneoftheabovemechanicalmethods.However,itshouldbenotedthatbacteria,virusorenvironmentalsamplescanbethesourceofproteinandthusWesternblottingisnotrestrictedtocellularstudiesonly.

Assorteddetergents,salts,andbuffersmaybeemployedtoencouragelysisofcellsandtosolubilizeproteins.Proteaseandphosphataseinhibitorsareoftenaddedtopreventthedigestionofthesamplebyitsownenzymes.Tissuepreparationisoftendoneatcoldtemperaturestoavoidproteindenaturing.

Acombinationofbiochemicalandmechanicaltechniques–includingvarioustypesoffiltrationandcentrifugation–canbeusedtoseparatedifferentcellcompartmentsandorganelles.

Gelelectrophoresis

Mainarticle:

Gelelectrophoresis

Theproteinsofthesampleareseparatedusinggelelectrophoresis.Separationofproteinsmaybebyisoelectricpoint（pI）,molecularweight,electriccharge,oracombinationofthesefactors.Thenatureoftheseparationdependsonthetreatmentofthesampleandthenatureofthegel.Thisisaveryusefulwaytodetermineaprotein.

Byfarthemostcommontypeofgelelectrophoresisemployspolyacrylamidegelsandbuffersloadedwithsodiumdodecylsulfate（SDS）.SDS-PAGE（SDSpolyacrylamidegelelectrophoresis）maintainspolypeptidesinadenaturedstateoncetheyhavebeentreatedwithstrongreducingagentstoremovesecondaryandtertiarystructure（e.g.disulfidebonds[S-S]tosulfhydrylgroups[SHandSH]）andthusallowsseparationofproteinsbytheirmolecularweight.SampledproteinsbecomecoveredinthenegativelychargedSDSandmovetothepositivelychargedelectrodethroughtheacrylamidemeshofthegel.Smallerproteinsmigratefasterthroughthismeshandtheproteinsarethusseparatedaccordingtosize（usuallymeasuredinkilodaltons,kDa）.Theconcentrationofacrylamidedeterminestheresolutionofthegel-thegreatertheacrylamideconcentrationthebettertheresolutionoflowermolecularweightproteins.Thelowertheacrylamideconcentrationthebettertheresolutionofhighermolecularweightproteins.Proteinstravelonlyinonedimensionalongthegelformostblots.

Samplesareloadedintowellsinthegel.Onelaneisusuallyreservedforamarkerorladder,acommerciallyavailablemixtureofproteinshavingdefinedmolecularweights,typicallystainedsoastoformvisible,colouredbands.Whenvoltageisappliedalongthegel,proteinsmigrateintoitatdifferentspeeds.Thesedifferentratesofadvancement（differentelectrophoreticmobilities）separateintobandswithineachlane.

Itisalsopossibletouseatwo-dimensional（2-D）gelwhichspreadstheproteinsfromasinglesampleoutintwodimensions.Proteinsareseparatedaccordingtoisoelectricpoint（pHatwhichtheyhaveneutralnetcharge）inthefirstdimension,andaccordingtotheirmolecularweightintheseconddimension.

Transfer

Inordertomaketheproteinsaccessibletoantibodydetection,theyaremovedfromwithinthegelontoamembranemadeofnitrocelluloseorpolyvinylidenedifluoride（PVDF）.Themembraneisplacedontopofthegel,andastackoffilterpapersplacedontopofthat.Theentirestackisplacedinabuffersolutionwhichmovesupthepaperbycapillaryaction,bringingtheproteinswithit.AnothermethodfortransferringtheproteinsiscalledelectroblottingandusesanelectriccurrenttopullproteinsfromthegelintothePVDFornitrocellulosemembrane.Theproteinsmovefromwithinthegelontothemembranewhilemaintainingtheorganizationtheyhadwithinthegel.Asaresultofthis"blotting"process,theproteinsareexposedonathinsurfacelayerfordetection（seebelow）.Bothvarietiesofmembranearechosenfortheirnon-specificproteinbindingproperties（i.e.bindsallproteinsequallywell）.Proteinbindingisbaseduponhydrophobicinteractions,aswellaschargedinteractionsbetweenthemembraneandprotein.NitrocellulosemembranesarecheaperthanPVDF,butarefarmorefragileanddonotstandupwelltorepeatedprobings.

TheuniformityandoveralleffectivenessoftransferofproteinfromthegeltothemembranecanbecheckedbystainingthemembranewithCoomassieBrilliantBlueorPonceauSdyes.PonceauSisthemorecommonofthetwo,duetoPonceauS'shighersensitivityanditswatersolubilitymakesiteasiertosubsequentlydestainandprobethemembraneasdescribedbelow.[6]

Blocking

Sincethemembranehasbeenchosenforitsabilitytobindproteinandasbothantibodiesandthetargetareproteins,stepsmustbetakentopreventinteractionsbetweenthemembraneandtheantibodyusedfordetectionofthetargetprotein.Blockingofnon-specificbindingisachievedbyplacingthemembraneinadilutesolutionofprotein-typically3-5%Bovineserumalbumin（BSA）ornon-fatdrymilk（bothareinexpensive）inTris-BufferedSaline（TBS）,withaminutepercentageofdetergentsuchasTween20orTritonX-100.Theproteininthedilutesolutionattachestothemembraneinallplaceswherethetargetproteinshavenotattached.Thus,whentheantibodyisadded,thereisnoroomonthemembraneforittoattachotherthanonthebindingsitesofthespecifictargetprotein.Thisreduces"noise"inthefinalproductoftheWesternblot,leadingtoclearerresults,andeliminatesfalsepositives.

Detection

Duringthedetectionprocessthemembraneis"probed"fortheproteinofinterestwithamodifiedantibodywhichislinkedtoareporterenzyme,whichwhenexposedtoanappropriatesubstratedrivesacolourimetricreactionandproducesacolour.Foravarietyofreasons,thistraditionallytakesplaceinatwo-stepprocess,althoughtherearenowone-stepdetectionmethodsavailableforcertainapplications.

Twosteps

∙Primaryantibody

Antibodiesaregeneratedwhenahostspeciesorimmunecellcultureisexposedtotheproteinofinterest（orapartthereof）.Normally,thisispartoftheimmuneresponse,whereasheretheyareharvestedandusedassensitiveandspecificdetectiontoolsthatbindtheproteindirectly.

Afterblocking,adilutesolutionofprimaryantibody（generallybetween0.5and5micrograms/mL）isincubatedwiththemembraneundergentleagitation.Typically,thesolutioniscomposedofbufferedsalinesolutionwithasmallpercentageofdetergent,andsometimeswithpowderedmilkorBSA.Theantibodysolutionandthemembranecanbesealedandincubatedtogetherforanywherefrom30minutestoovernight.Itcanalsobeincubatedatdifferenttemperatures,withwarmertemperaturesbeingassociatedwithmorebinding,bothspecific（tothetargetprotein,the"signal"）andnon-specific（"noise"）.

∙Secondaryantibody

Afterrinsingthemembranetoremoveunboundprimaryantibody,themembraneisexposedtoanotherantibody,directedataspecies-specificportionoftheprimaryantibody.Antibodiescomefromanimalsources（oranimalsourcedhybridomacultures）;ananti-mousesecondarywillbindtoalmostanymouse-sourcedprimaryantibody,whichallowssomecostsavingsbyallowinganentirelabtoshareasinglesourceofmass-producedantibody,andprovidesfarmoreconsistentresults.Thisisknownasasecondaryantibody,andduetoitstargetingproperties,tendstobereferredtoas"anti-mouse,""anti-goat,"etc.Thesecondaryantibodyisusuallylinkedtobiotinortoareporterenzymesuchasalkalinephosphataseorhorseradishperoxidase.Thismeansthatseveralsecondaryan