1.Preparethetransferbuffer. 2.Followingelectrophoresis,equilibratethegelsintransferbuffer.Equilibrationfacilitatestheremovalofelectrophoresisbuffersaltsanddetergents.Thelengthoftimerequiredforequilibrationisdependentonthegelthickness.Forexample,15min.fora0.75mmSDS-PAGEgel. 3.Cutthemembranetothedimensionsofthegel.Wetthemembranebyslowlyslidingitata45°angleintotransferbufferandallowingittosoakfor15-30min. 4.Cutfilterpapertothedimensionsofthegel.Twopiecesofthickfilterpaper(orsixpiecesofthinfilterpaper)pergelareneededforeachgel/membranesandwich.Completelysaturatethefilterpaperbysoakingintransferbuffer. 5.Ifmorethanonefull-sizegelistobetransferredatonetime,cutapieceofdialysismembranewiththeappropriatemolecularweightcutofftothedimensionsofthegel.Completelywetthedialysismembraneintransferbuffer. AssemblyoftheUnitforStandardTransfers(Wearglovesforthisproceduretoavoidcontaminationofmembranes.) 1.Removethesafetycoverandthestainlesssteelcathodeassembly. 2.Placethepre-soakedsheetoffilterpaperontotheplatinumanode.Rollapipetortesttubeoverthesurfaceofthefilterpaper(likearollingpin)toexcludeallairbubbles.Ifthinfilterpaperisused,repeatwithtwomoresheetsofbuffer-soakedfilterpaper. 3.Placethepre-wettedblottingmediaontopofthefilterpaper.Rolloutallairbubbles. 4.Carefullyplacetheequilibratedgelontopofthetransfermembrane,aligningthegelonthecenterofthemembrane.Transferwillbeincompleteifanyportionofthegelisoutsidetheblottingmedia.Rolloutallairbubbles. 5.Placeanothersheetofpre-soakedfilterpaperontopofthegel,carefullyremovingairbubblesfrombetweenthegelandfilterpaper.Ifthinfilterpaperisused,placethreesheetsontopofthegel,andremovebubblesfrombetweeneachlayer. 6.Ifmorethanonefull-sizegelistobetransferred,placesheetofpre-soakeddialysismembraneontopofthefilterpaperstack.Repeattheprocedurefromstep2.Uptofourminigelscanbetransferredatthesametimebyplacingthemside-by-sideontheanodeplatform. 7.Carefullyplacethecathodeontothestack.Presstoengagethelatcheswiththeguidepostswithoutdisturbingthefilterpaperstack. 8.Placethesafetycoverontheunit.Plugtheunitintothepowersupply.Normaltransferpolarityiscathodetoanode,i.e.,redwiretotheredoutletandblackwiretoblackoutletonthepowersupply. Caution:Donotreversepolarity.Thiswillresultindamagetothestainlesssteelcathode. 9.Turnonthepowersupply.Transferminigelsfor15-30min.at10-15V.Largegelscanbetransferredfor30min.to1hr.at15-25V.Donotexceed25Vwiththisinstrument.Acurrentlimit(3mA/cm2forlargegels;5.5mA/cm2forminigels)isrecommendedtopreventexcessiveheatingduringtherun.Underthestrongfieldsdevelopedbythisapparatus,transfersmaynotalwaysbequantitative.Acertainquantityofproteinmaybetransferredthroughthemembraneandontothefilterpaperbelow.(i.e.Constantcurrent~15QmMfor2minigels) 10.Followingtransfer,turnthepowersupplyoff,anddisconnecttheunitfromthepowersupply.Removethesafetycoverandthecathodeassembly.Discardthefilterpaper(anddialysismembrane,ifused).ThetransferefficiencycanbemonitoredbystainingthegelwithCoomassieblueR-250proteinstainorwithBio-Rad’sSilverStainKit.Alternatively,prestainedmolecularweightstandardscanbeused,oraportionofthemembranecanbestainedfortotalproteinwithcolloidalgold,BiotinBlotTotalProteinStain,orananionicdyesuchasAmidoBlack.Zeta-ProbemembranecanbestainedwiththeBiotin-BlotTotalProteinStain. SDSmaybeaddedtoBuffer1toincreaseproteinelutionfromthegel: 48mMTris,39mMglycine,(20%methanol),1.3mMSDS(0.0375%),pH9.2.Dissolve5.82gTrisand2.93gglycine,and0.0375gSDSor3.75mlof10%SDSinddH2O(add200mlofmethanol);adjustthevolumeto1literwithdiluteddH2O. DONOTADDACIDORBASETOADJUSTpH. TowbintransferbufferforSDS-proteinsusingnitrocellulose(withmethanol)orZeta-Probemembrane(withoutmethanol): 25mMTris,192mMglycine(20%methanol),pH8.3Dissolve3.03gTrisand14.4gglycineinddH2O(add200mlofmethanol);adjustvolumeto1literwithddH2O. DONOTADDACIDORBASETOADJUSTpH.
