WESTERNBLOTPROTOCOLInaWesternblot,proteinsthatareseparatedonpolyacrylamidegelsonthebasisofsizearetransferredtoamembranefordetectionwithantibodies.PreparationofHumanCellExtracts(perYasuItoh,5/91)1.Add2volumesofautoclavedH2Oforeachvolumeofcellpellet.2.Bringto1mMPMSF.3.Sonicateonice(1sec/ml,5timesatsetting5).Wait30secondsbetweeneachsonication.4.Pelletdebrisinthemicrocentrifugeat13,000RPMfor5minutes.5.DilutewithwatertoOD280=3.0.Storeat-20°C.Thesestocksshouldbegoodforatleastoneyear.PreparationofBacteriallyExpressedProteins1.Growa10mlbacterialculturetosaturationwiththeappropriateantibiotic.IfIPTGinductionisneeded,transfer200µlofthesaturatedcultureto10mlofLbrothwithantibiotic.IPTGmaybeaddedatthistime,orthecellsmaybegrowntoattainexponentialgrowthbeforeaddingtheIPTG.Add10-100µlof1MIPTGsothatthefinalconcentrationofIPTGisfrom1-10mM.Induceproteinsynthesisfor1-3hours.ThefinalyieldofproteinmaybeaffectedbytheconcentrationofIPTG,theinductiontime,andthebacterialconcentration.2.Centrifugeat2,500RPMfor15minutesat4°C.Discardthesupernatant.3.Dissolvethepelletin1mloflysatebuffer.Bringto1mMPMSFbyadding100mMPMSFstock(17.42mg/mlinmethanol).Thissolutionmaybekeptfor1weekat-20°C.4.Add50µlofa10mg/mllysozymesolutionmadein0.25MTris-HCl,pH8.Makethelysozymesolutionfresheachtime.Donotvortextheenzyme.Shakethecellsonicefor15minutes.5.Transferthecellsolutiontoa4mlFalcontubeandsonicateatsetting"4"forabout10secondsonice.6.Transferthislysatetomicrofugetubesandcentrifugefor15minutesonhighspeed(13,000rpm)at4°C.7.Atthispoint,youwillneedtodetermineiftheantigenisinthesupernatantorinthepelletusingaWesternblotorELISA.(Inthecaseoftherecombinant52kDRo/SSA,theantigenicpelletmaybestoredat-20°Cforabout1monthor-70°Cforabout3months.)SDS-PAGESeparatingGels(Hoefferbabygels):1.For2gels,mixinanErlenmeyerflaskinthefumehoodwithconstantstirringundervacuum(toremoveairbubbles)for15minutes:6mllowergelbufferatroomtemperature(duetotheSDS)6ml40%acrylamide12mldoubledistilledH20(--oruse7.95mlof30%acrylamideand10.05mlH20--)Increasethepercentageofacrylamidetoanalyzelowermolecularweightproducts(lessthan30kD),butcontinuetousea10%stackinggel.2.Assemblethegelholder.Wearglovestocleantheplateswithmethanolor95%ethanol.DrywithKimwipes.Assembleinagelcasterinthefollowingorder(for2gels):a).waxpaperb).thickplasticplatec).thin,flexIBLeplasticspacerplated).whiteglassplatee).Grayplasticsidespacerf).Clearglassplateg).thin,flexibleplasticspacerplateh).Repeat4-7,thenclampsides.3.Add0.12mlof10%ammoniumpersulfatesolutionand12µlofTEMED(keptat4°C)totheacrylamidesolutionpreparedabove.TheAPSsolutionshouldnotbemorethan1weekold.4.Pourtheacrylamidesolutionintothegelholdertoabout1cmabovetheblackmark.Thisblackmarkis3cmfromthetopoftheplasticholder.Add1/2PasteurpipetfullofH20saturatedbutanoltoeachplate.5.Waitatleast45minutesforpolymerizationthenrinsethetopofthegelswithdistilledH20.Thesegelsmaybestoredatroomtemperatureforabout1weekinasealedcontainerwithH20soakedtowelstoavoiddessicationofthegels.6.ClampinelectrophoresisholderandrinseagainwithH2O,thenblotthetopofthegelwithfilterpapertoremoveexcessH2O.Prepare10%SDSStacking(orupper)Gel:1.25mluppergelbufferatroomtemperature0.56ml40%acrylamide3.2mlH2O(--or0.75ml30%acrylamideplus3.0mlH2O--)30µl10%APSsolution10µlTEMEDLoadtheuppergelwithaPasteurpipetandaddthegelcomb.Watchpolymerizationaboutevery3minutestoseeifmoreuppergelsolutionneedstobeadded.Itshouldbecompletelypolymerizedwithin15minutes.RemovecombandaddTankBuffertothetopwithafunnel.Removeanybubblesatthebottomofthegel.Rinseallwellswithtankbuffer.PreparationofSample:1.Considervolumestobeloaded:Forthe1wellcomb,load10µlofproteinmolecularweightMarkersand500µlofsample.Use30µlofsampleperwellforthe10wellcomb.IfasimilargelispreparedforCoomassieblueorsilverstaining,additionalproteinneedstobeloadedrelativetothatusedforWesternblots.SeetheCoomasieandSilverStainingprotocolfordetails.2.Addsamplebuffertothesamples:*MolecularweightmarkersfromPromegaareat0.5mg/ml.Thesearediluted1:2inH2Oandstoredat-20°C.Beforeuse,add20µlofthisdilutionto10µlofH20and10µlof4xsamplebuffer(or20µlof2xsamplebuffer).Molecularweightsarelistedbelow.*Forhumancellextractsadd167µlof4xsamplebufferto500µlofMolt-4extract(A280=3.0)beforeheating.Thiswilldilutethesamplebuffertoa1xconcentration.*Forbacterialcellextracts,dissolvethefinalantigenicpellet(orheatthebacteria)in500µlof2xsamplebuffer,thendilute1:10inadditional2xsamplebuffer.*Forinsectcellsinfectedwithbaculovirus,usealysatefrom3000cellsperlaneina10wellcombwithdilutedsamplebuffer.#Heatsamplesat95°Cfor10minutesbeforeloADIngonthegel.ElectrophoreticSeparationofProteins:For1gel,electrophoreseinitiallyat20mAconstantcurrent.Whenthedyepassedthroughthestackinggel,increaseto30mA.Continueelectrophoresisuntilthedyehasrunoffbottomofthegel(lessthan2hours).Alternatively,anovernightelectrophoresismaybeperformedbyrunningthegeluntilthedyeishalfthroughthegelontheabovesettings,andthendecreasingthepowerto5mAovernight.Inthelattercase,the31kDmolecularweightmarkershouldbeatthebottomofthegelinthemorning.Disassembletheapparatus,anddiscardthestackinggel.Notchthebottomrightcornersoftheseparatinggelandaprecutnitrocellulosemembrane.Placethemembraneontopofthegel,andpeelthegeloffoftheplate.For2gels,followaboveprocedurebutdoublethecurrentsettings.ElectrophoreticTransferforDryBlots(Millipore):PrecutthreepiecesofWhatman3MMfilterpapertothesizeofthegel.SaturateonefilterwithworkingsolutionsA1,A2,orC,respectively.Onthebottomelectrode,placeinthefollowingorderfilterA1,thenfilterA2,thenitrocellulosemembrane,thepolyacrylamidegel,andfinallyfilterC.Removeanybubblesaftereachstep.Placethetopelectrodeonthestack.Begintransferat10-12constantvolts.Regulatevoltagetokeepthecurrentat105mA.Donotallowvoltagetoexceed15V.Theminimumtransfertimeis40minutes,themaximumis60minutes.Youwillobtainpoortransferifmorethan60minutesisused.ElectrophoreticTransferForWetBlots:1.Soakthegel(s)andthenitrocellulosemembrane(s)inwetblottransferbufferforaminimumof15minutestoremoveelectrophoresissaltsanddetergents.2.Foreachgel,saturatetwofiberpadsandtwoprecutWhatman3MMfilterpapersintransferbuffer.3.Assembleonthegraysideofacassetteinthefollowingorder:1fiberpad1Whatman3MMfilterpaperGelNitrocellulosemembrane1Whatman3MMfilterpaper1fiberpad4.Repeatwithasecondcassetteforthesecondgel,ifneeded.5.Insertthecassetteintotheelectrodemodule.Besuretocheckthedirectionsothatthetransferisfromthegeltothemembrane.Thegraysideofthecassetteshouldbenexttothegraysideoftheelectrodemodule(i.e.,thecathodeside).6.PlaceastirbarandaBio-Icecoolingunit(storedat-20°C)inthebuffertank.Placetheelectrodemoduleinthebuffertank.7.Fillthetankwithwetblottransferbuffertothebottomedgeofthetoprowofholesonthecassette.Placethebuffertankonamagneticstirplateandstiratmediumspeed.8.Attachelectrodesandelectrophoreseat100Vfor1hour,or30Vovernight.DetectionofAntigenonMembranes:1.Stainthemembranewith0.1%fastgreenfor1minute.RinsefourtimeswithH20.CutawaythemolecularweightmarkerlaneandshakeitinTBSTfor30minutes(orlonger).Therestofthemembranemaybeslicedinstrips,ifneeded.Fora10wellcomb,eachwellmaybecurinhalf.Notchtheleftbottomedgeofallstripsfororientationpurposes.2.Blockthemembranesina5%non-fatmilk-TBSTsolutionforatleast30minutes(overnightisfine)whileshaking.For3mmstripsuseabout1mlofTBST-milk.MembranesmaybestoredinTBSTat4°Cforupto1week.3.Removetheblockingsolution.RinsemembranesinTBSTonce.4.Add500µlforthinsections,or5mlforanentiremembrane,ofserumdilutedin5%milk-TBST.Atypicalserumdilutionis10-2.Incubateatroomtemperaturefor30minuteswhileshaking.5.Wash4timesforfiveminutesperwashinTBSTatroomtemperature.6.Add500µlofdilutedalkaline-phosphataseconjugatedsecondaryantibodyandincubatefor30minutesatroomtemperaturewithshaking.(Sigmaanti-humanIgGconjugateisdiluted1:2000in5%milk-TBST).7.Wash4timesforfiveminutesperwashwithTBSTatroomtemperature.8.Add500µlofBCIP/NBTsubstrate(KPLinbrownbottle,storedat4°C).Thissolutionshouldbeyellow,notbrown.Waitabout15minutesforbandstoappear.Watchcarefullysincethereactiontimeswillvary.Rinsewithwatertostopdeveloping.Whilethemembranesaredeveloping,stainthemolecularweightmarkerlanewith0.1%AmidoBlacksolutionfor1minute,removethismembraneandimmediatelyaddtoAmidoBlackdestainforabout10seconds(longertimesresultinshrinkageofthemembrane)beforerinsingextensivelyinH20.PhotographthemembraneswithPolaroidType57film.Focuswiththeoverheadandfocusinglightson,andtheshutterandf-stopwideopen("B"andf4.5).Closetheshutter.Photographusingonlytheroomlights(focusinglightsoff)atf8,1/60secondexposure.RECIPESLysateBuffer(PlasmidPrepSolution#1:0.1MNaCl,50mMTris-HClpH7.8-8.5,10mMEDTA)For100ml,add2mlof5MNaCl,5mlof1MTris-HClstockattheappropriatepH,2ml0.5MEDTAandQ.S.withdistilledwater.Autoclave.5%NonfatDryMilkPrepareusingCarnationNonfatDryMilkinTBST.10%AmmoniumPersulfate(APS)Solution0.12gAPS1.2mlH2O)Keepfornomorethanweekweekintherefrigerator.TBST(10mMTris-HCl,0.15MNaCl,8mMsodiumazide,0.05%tween-20,pH8.0)ChemicalFor4litersFor500mlTris4.84g0.61gTrisNaCl35.06g4.38gNaClNaN32.0g500µl10%NaN3Tween-202.0ml250µlTween-20AdjustpHto8.0withHCl.QSwithwaterandstoreat4°C.TankBuffer(25mMTris-HCl,0.2Mglycine,0.35%SDS)ChemicalFor4litersFor500mlTris12.11g1.51gTrisGlycine57.6g7.2gSDS4.0g17.5ml10%SDSQSwithH2O.pHshouldbe8.3butadjustingisnotneeded.Storeat4°CTransferBuffers:Wetblottransferbuffer(3L)(25mMTris-HCl,0.2Mglycine,20%methanol).9.08gTris43.24gglycine600mlmethanolQSto3Lwithwater.DryblottransferbuffersStockSolutionA1(3MTris)36.3gTris,dissolvedindoubledistilledwaterandQSto100mlStoreatroomtemperature.StockSolutionA2(0.3MTris)3.63gTris,dissolvedindoubledistilledwaterandQSto100mlStoreat4°C.StockSolutionC(0.3MTris,0.4Maminohexane)3.63gTris,5.2gaminohexane,dissolvedindoubledistilledwaterandqstoto100ml.Storeat4°C.DryBlotWorkingSolutions(madefromfryblottransferbuffers)7mlH202mlMethanol1mlstocksolutionofA1,orA2orCStoreat4°C.LowerGelBuffer(1.5MTris-HCl,0.4%SDS,pH8.8)For100ml:18.17gTris4ml10%SDSAdjustpHto8.8withHCl.QSto100mlwithwater.Storeatroomtemperature.UpperGelBuffer0.5MTris-HCl,0.4%SDS,pH6.8)For100ml:6.06gTris4ml10%SDSAdjustpHto6.8withHCl.QSto100mlwithwater.Storeatroomtemperature.4xSampleBuffer(madefromstocksamplebuffer)(0.24MTris-HCl,0.24MSDS,40%glycerol,20%2-mercaptoethanol,pH6.8)8mlofstocksamplebuffer2mlofconcentrated2-mercaptoethanolTransferto1mlaliquotsandstoreat-20°C.2xSampleBuffer(HoefferScientific)(125mMTris-HCl,4%SDS,20%glycerol,10%2-mercaptoethanol,0.004%bromophenolblue).Prepareinfumehood!2.5ml0.5MTris-HCl,pH6.8and4.0mlof10%SDS(--or--2.5mlofuppergelbufferand3.6mlof10%SDS)2.0mlglycerol1.0mlconcentrated2-mercaptoethanol0.4mgbromophenolblue(youmayuselessbutdon"taddmorethan0.4mg)QSto10mlwithwater.Transferto1mlaliquotsandstoreat-20°C.StockSampleBuffer(fromCraigWasson)(0.312MTris-HCl,0.346MSDS,50%glycerol,pH6.8)3.03gTris8.0gSDS40mlglycerolAdjustpHto6.8withHCl.QSto80mlwithwater.Storeatroomtemperature.0.1%FastGreen(in10%aceticacidand20%methanol)0.2gFastGreen20mlaceticacid40mlmethanol140mldoubledistilledwater.0.1%AmidoBlack(in10%aceticacidand45%methanol)0.2gBuffaloBlack90mlmethanol20mlaceticacid90mldoubledistilledwater.AmidoBlackDestain(2%aceticacid,45%methanol)225mlmethanol10mlaceticacidQSto500mlwithdoubledistilledwater.