western blotting 2.docx

western blotting 2.docx

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westernblotting2

Westernblotting

BackgroundandIntroduction

◆Background

Afterelectrophoresis,onlyaminuteamountofproteinornucleicacidispresentinbandsonelectropherograms.Inspiteofthis,thereisoftenaneedtoextractthedesiredbiomoleculefromthegelforfurtherinvestigation.Thissometimesinvolvesthetediousandcumbersomeprocessofcrushingslicesofthegelinabuffertoreleasethetrappedproteinsornucleicacids.Techniquesarenowavailableforremovingnucleicacidsandproteinsfromgelsandcharacterizingthemusingprobestodetectcertainstructuralfeaturesorfunctions.Afterelectrophoresis,thebiomoleculesaretransferredor“blotted”outofthegeolontoanitrocellulosefilterornylonmembrane.Thedesiredbiomoleculeisnowaccessibleonthefilterforfurtheranalysis.ThefirstblottingtechniquewasreportedbyE.Southernin1975.UsinglabelledcomplementaryDNAprobes,hesearchedforcertainnucleotidesequencesamongDNAmoleculesblottedfromthegel.ThistechniqueofdetectingDNA-DNAhybridizationiscalledSouthernblotting.ThegeneralblottingtechniquehasnowbeenextendedtothetransferanddetectionofspecificRNAwithlabelledcomplementaryDNAprobes（Northernblotting）andthetransferanddetectionofproteinsthatreactwithspecificantibodies（Westernblotting）.Inpractice,theelectropherogramisalkalitreated,neutralized,andplacedincontactwiththefilterornylonmembrane.Abufferisusedtofacilitatethetransfer.Thelocationofthedesirednucleicacidorproteinisthendetectedbyincubationofthemembranewitharadiolabelledprobeandautoradiography,byuseofabiotinylatedprobeorbylinkagetoanenzyme-catalyzedreactionthatgeneratesacolour.

Blottingtechniqueshavemanyapplications,includingmappingthegenesresponsibleforinheriteddiseasesbyusingrestrictionfragmentlengthpolymorphisms（PFLPs）,screeningcollectionsofclonedDNAfragments（DNAlibraries）,“DNAfingerprinting”foranalysisofbiologicalmaterialremainingattheidentificationofspecificproteins.

Inthisexperiment,westernblottingisemployedtoidentifythespecificproteinontheelectrophoresisgel.

◆Introduction

Inexperimentfour,wehavetriedthetechniqueofSDS-PAGE.SodiumDodecylSulfate-PolyacrylamideGelElectrophoresis（SDS-PAGE）istheelectrophoretictechniquesappliedtothemeasurementofthemolecularweightsofbiologicalmolecules.Iftheproteinsamplesaretreatedsothattheyhaveauniformcharge,electrophoreticmobilitythendependsprimarilyonsize.Themolecularweightsofproteinsmaybeestimatediftheyaresubjectedtoelectrophoresisinthepresenceofadetergent,sodiumdodecylsulphate（SDS）,andadisulfidebondreducingagent,mercaptoethanol.Thismethodisoftencalled“denaturingelectrophoresis.”.SDS-PAGEisvaluableforestimatingthemolecularweightofproteinsubunits.Thismodificationofgelelectrophoresisfindsitsgreatestuseincharacteringthesizesanddifferenttypesofsubunitsinoligomericproteins.SDS-PAGEislimitedtoamolecularweightrangeof10,000to200,000.Gelsoflessthan2.5%acrylamidemustbeusedfordeterminingmolecularweightsabove200,000,butthesegelsdonotsetwellandareveryfragilebecauseofminimalcross-linking.Amodificationusinggelsofagarose–acrylamidemixturesallowsthemeasurementofmolecularweightsabove200,000.

InSDS-PAGE,afterrunningandstainingwiththedyeCoomassieBlue,deeplycoloredbandsappearedonthegelwherevertherewasaprotein.Ifmolecularweightstandardswereincludedonthegel,itisfeasibletoestimatethemolecularweightforaspecificproteinpresentonthegel.SDS-PAGEisindeedaveryeffectiveanalyticaltooltoachievefractionationofproteinmixtures,toanalyzepurity,andtoestimatemolecularweight,butitprovidesnoexperimentaldatatoprovetheidentityofanyofthedyedproteinbands.ACoomassieBluestainsimplyindicatesthepresenceandlocationofeachandeveryproteinonthegel.Itisoftenpossibletoidentifyproteinsbytreatinggelbandsdirectlywithchemicalreagentsthatreactwithaspecificprotein.Forexample,theidentityandlocationofanenzymemaybenotedbytreatingthegelwithasubstratethatisconvertedtoacolouredproductbyenzyme.However,proteinsaredeeplyembeddedinthepolyacrylamidegelmatrixandarenotreadilyaccessibletomostanalyticalreagents.Thishindersspecificanalysisoftheproteinbandsinordertoidentifyindividualproteins.ProteinsseparatedbyPAGEmaybetransferred（orblotted）fromthegeltoathinsupportmatrix,usuallyanitrocellulosemembrane,whichstronglybindsandimmobilizesproteins.Theproteinblotsonthemembranesurfacearemoreaccessibletochemicalorbiochemicalreagentsforfurtheranalysis.Whenthetransferprocessiscoupledwithproteinidentificationusinghighlyspecificandsensitiveimmunologicaldetectiontechniques,theprocedureisWesternBlotting.Westernblottingorimmunoblottingassaysofproteinshavemanyadvantagesincludingtheneedforonlysmallreagentvolumes,shortprocessingtimes,relativelyinexpensiveequipment,andeaseofperformance.

TobegintheWesternblotprocedure,aproteinmixtureforanalysisandfurthercharacterizationisfractionatedbyPAGE.Sincedenaturing,SDS-PAGEresultsinbetterresolutionthanPAGEperformedundernativeconditions,SDS-PAGEisusuallypreferred;however,thedetectionmethodusedattheconclusionoftheblottingexperimentmustbeabletorecognizedenaturedproteinsubunits.Thenextstepinvolvesselectionofthemembranematrixfortransfer.Threetypesofsupportmatricesareavailableforuse:

nitrocellulose,nylon,andpolyvinyl-difluoride（PVDF）.Nitrocellulosemembranes,currentlythemostwidelyusedsupports,haveasatisfactoryproteinbindingcapacity（100μg/cm2）,buttheydisplayweakbindingofproteinsofmolecularweightssmallerthan14,000andtheyaresubjecttotearing.Bindingofproteinstonitrocellulosemembranesisnoncovalent,mostlikelyhydrophobic.Nylonmembranesarestrongerthannitrocelluloseandsomehaveabindingcapacityupto450μg/cm2.However,sincetheyarecationic,theyonlyweaklybindbasicproteins.Duringdetectionprocedures,nylonmembraneoftendisplayhighbackgroundcolours,soitisdifficulttovisualizeproteinsofinterest.PVDFmembranesbindproteinsstrongly（125μg/cm2）and,becauseoftheirhydrophobicnature,givelightbackgroundcolourafteranalysis.Foroverallgeneraluseinproteintransferandimmunoblotting,nitrocellulosemembranesarethemostcommonchoice,aswedidinthisexperiment.

Theactualblottingprocesscanbeaccomplishedbyoneofthetwomethodsbelow:

passive（orcapillary）transferandelectroblotting.Inpassivetransfer,themembraneisplacedindirectcontractwiththepolyacrylamidegelandorganizedinasandwich-likearrangementconsistingof（frombottomtotop）filterpapersoakedwithtransferbuffer,gel,membrane,andmorefilterpaper.Thesandwichiscompressedbyaheavyweight.Bufferpassesbycapillaryactionfromthebottomfilterpaperthroughthegel,transferringtheproteinmoleculestothemembrane,wherethemacromoleculesareimmobilized.Passivetransferisverytimeconsuming,sometimesrequiring1-2daysforcompleteproteintransfer.Fasterandmoreefficienttransferisaffordedbytheuseofanelectroblotter.Hereasandwichoffilterpaper,gel,membrane,andmorefilterpaperispreparedinacassette,whichisplacedbetweenplatinumelectrodes.Anelectriccurrentispassedthroughthegel,causingtheproteinstoelectrophoreseoutofthegelandontothemembrane.

Thus,theWesternblotprocedureisconcludedbyprobingtheblottedproteinbandsanddetectingaspecificproteinorgroupofproteinsamongtheblots.Inotherwords,visualizationofspecificproteinblotsmustbepossible.Themostspecificidentificationtechniquesarebasedonimmunology（antigen-antibody）interactions.Ageneralprocedureforimmunoblottingisoutlinedhere:

（1）.Proteinsaretransferredfromelectrophoresisgeltonitrocellulosemembrane.Blockerproteinsbindtounoccupiedsitesonthemembrane.

（2）.Themembraneisincubatedwithaprimaryantibodydirectedagainsttheproteinofinterest.

（3）.Asecondaryantibodyisdirectedagainsttheprimaryantibody.

（4）.Thesecondantibodyisconjugatedwithanenzymetoprovideadetectionmechanism.Substratesolutionisaddedtotheblot.Theconjugatedenzymecatalyzestheconversionofsubstratetoproducttoformacoloredprecipitateatthesiteoftheprotein-antibodycomplex.

Beforetheproteindetectionprocesscanbegin,itisnecessarytoblockproteinbindingsitesonthemembranethatarenotoccupiedbyblottedproteins.Thisisessentialbecauseantibodiesusedtodetectblottedproteinsarealsoproteinsandwillbindtothestillremainingonblottedmembraneandinterferewithdetectionprocedures.Proteinbindingsitesstillremainingonblottedmembranesmaybeblockedbytreatmentwithsolutionsofcasein（majorproteininmilk）,gelation,orbovineserumalbumin.Inthisexperiment,bovineserumalbuminisusedasblocker.

Theblottedmembrane,withallproteinbindingsitesoccupied,canmowbetreatedwithanalyticalreagentsfordetectionofspecificproteins.Typically,theblottedmembraneisincubatedwithanantibodyspecificfortheproteinofinterest.Thisistheprimaryantibody,whichisaproteinoftheimmunoglobulinG（IgG）class.Theprimaryantibodybindstothedesiredprotein,forminganantigen-antibodycomplex.Theinteractionbetweentheproteinanditsantibodydosenotusuallyresultinavisiblesignal.Theblotisthenincubatedwithasecondaryantibody,whichisdirectedagains