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