WesternblottingWord文件下载.docx
- 文档编号:6819309
- 上传时间:2023-05-07
- 格式:DOCX
- 页数:15
- 大小:46.92KB
WesternblottingWord文件下载.docx
《WesternblottingWord文件下载.docx》由会员分享,可在线阅读,更多相关《WesternblottingWord文件下载.docx(15页珍藏版)》请在冰点文库上搜索。
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,whichisdirectedagainstt
- 配套讲稿:
如PPT文件的首页显示word图标,表示该PPT已包含配套word讲稿。双击word图标可打开word文档。
- 特殊限制:
部分文档作品中含有的国旗、国徽等图片,仅作为作品整体效果示例展示,禁止商用。设计者仅对作品中独创性部分享有著作权。
- 关 键 词:
- Westernblotting