Western Blotting (or Immunoblotting)
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1 SelimeAksit/LIULAB LastRevised:6/22/10 Protocol Original6/8/10 WesternBlotting(orImmunoblotting) Buffers&Reagentstoprepare: 10%APS(w/v)[make~5mLatatime]: For5mL: Volume Material 0.5g 5mL AmmoniumPersulfate MQ H 2 O F/S,storeat4 o C 1XM9LiquidMedia+0.4%Sugar(F/S) For400mLofMannitol For400mLofGlucose Volume Material Volume Material 80mL 16mL 304mL 5XM9 10%Mannitol MQ H 2 O 80mL 8mL 312mL 5XM9 20%Glucose MQ H 2 O F/SstoreatRoomTemp SDS PAGE5xSampleBuffer: Material 250mM TrisBufferpH6.8 10% SDS 50% Glycerol 0.5% Bromophenolblue storeatroomtemp;add10%β Mercaptoethanoltosmallvolume of samplebufferwhenneeded beforeloadingsamples SDS PAGETransferBuffer: For1Liter: Volume Material 3.0g TrisBase 14.5g Glycine 200mL Methanol Bringto1LwithMQ H 2 O Addallto1000mLglassflask,storeat4 o C 10XProteinElectrophoresisBuffer: For500mLs Volume Material 5.0g 15.0g 72.0g SDS TrisBase Glycine Bringto500mLwithMQ H 2 O Abbreviations: w/v=weight/volume F/S=filter/sterilized SDS=SodiumDodecylsulfate PAGE=polyacrylamidegel electrophoresis 10 =10minutes F/SstoreatRoomTemp 1
2 SelimeAksit/LIULAB LastRevised:6/22/10 Protocol Original6/8/10 Addallto500mLglassflask,storeatRT Diluteto1XProteinElectrophoresisBufferforuse. WesternBlotProtocol: GelPrep(Day1) Weargogglesinthecaseofsplashingoftoxicreagents! Washglassplates,castingframe,andcomb;thendry! 1. Prepare10%SDS PAGEGel: Volume Material 2.5mL 1.5MTrisBufferpH µL 10%SDS(w/v) 3.35mL PROTOGELacrylamide*neurotoxin 4.1mL MQ H 2 O *STOP Prepareapparatus:#1athen1binFigureabove 50µL 10%(w/v)APSF/S(4 o C)*shouldbeusedw/in1month 5µL TEMED(stinky) Mixbygentlyswirlingallthecontentsina50mLbeaker 2. Pourgelbetweentheglassplatessetup(Figure#1b)tillalittlebelowwherethecombwouldbe. 3. Flattenthegelimmediatelywith100µLofn butanol 4. Solidifygel Removen butanolusingpapertowel.rinsethe3xwith600µlmq H 2 O,removew/papertowel. 6. Preparestackinggel: Volume Material 2
3 SelimeAksit/LIULAB LastRevised:6/22/10 Protocol Original6/8/ mL 50µL 0.690mL 3.6mL *STOP 25µL 5µL 1MTrisBufferpH6.8 10%SDS(w/v) PROTOGELacrylamide*neurotoxin MQ H 2 O Checkapparatusbeforecontinuingtoadd 10%(w/v)APSF/S(4 o C)*shouldbeusedw/in1month TEMED(stinky) Mixallthecontentsgentlyina50mLbeakerbeforeaddingtoSDSgel 7. PourstackinggelontopoftheSDS PAGEgel,placeincombslowlyandinstraight DONOTMAKE AIRBUBBLES! 8. ADDCOMB&DONOTREMOVEIT. 9. Solidifystackinggelw/comb Oncesolidified(checkbeaker),ripoutalargepieceofplasticwrap.Placetwopapertowelsonthe plasticwrap.soakthepapertowelswithjustenough1xproteinelectrophoresisbuffer.takeplates withcombandgelstillinbetweenandwrapitinthewetpapertowelstightly.thenwraptheplastic wrapcompletelyaroundeverything. 11. Storethewrappedgelintherefrigerator(4 o C)overnight shorterplatefacingupwards. 3
4 SelimeAksit/LIULAB LastRevised:6/22/10 Protocol Original6/8/10 RunningWesternBlot:Electrophoresis(Day2) 1. Preparethesamplebufferbyadding1/10volumeβ Mercaptoethanol. 2. Add~1/4 1/2volumeofsamplebuffertosample.Example:10µLofsample+5µLofβ MeSmpleBfr 3. Incubatetubesat95 o C,10.Spinbrieflyinpicofugefor2 3seconds. 4. Assembleapparatus(#2inFigure).Unwrapgel,wipeoffexcessresidue.Tightlysealgel/plateswith combfacingtowardtheinsideandthebufferdambyclampingthegreensidesofclampingframe (#2).Thenplaceentireelectrodeassemblyintoclearbox(#3 withoutgreencap). 5. Pour1XProteinElectrophoresisBufferinbetweentheelectrodeassemblytothetop.Checkifthere areanyleaksinthe reservoir betweenthesealedgelanddam.iftherearenoleaks,filltherestof theclearboxwith1xproteinelectrophoresisbufferuntilthe 2gel linemark. 6. Removecomb.Loadsamples. 7. Putongreencap Matchblacktoblack;andredtored.Turnonthemachine.Havethewires pluggedinblacktoblack;andredtored. 8. Runconstantvoltage:200V, Emptyoutthe1XProteinElectrophoresisBufferintothesink. RunningWesternBlot:Transfer 1. Gettwofilterpapers,cutapieceofmembrane(slitthetopleftcornerofthemembrane). 2. Soakthefilterpapers,membraneandspongesinalittlebitoftransferbuffer5 beforeconducting thetransfer. 3. Preparethetransfersandwich:black sponge filterpaper gel membrane(makesuretomatch thetopleftcornerslitofthegelwiththeslitofthemembrane) filterpaper sponge clear: CLAMP 4. Placethesandwichintotheblack and redbox.note:theblacksideofthesandwichshouldfacethe blacksideofthered and blackbox. 5. Placetheblackandredboxwithsandwichintotheclearbox(Figure #3) Makesurethatblacksideismatchedwiththeblackoftheclearbox.Redwithred. 6. Putinstirbarandicebox.Placeclearboxonstirmachine. 7. Fillupboxwith1XTransferBuffer(retrievedfromthefridge4 o C)until BlottingLine. 8. Coverwithgreencaponclearbox;blackmatcheswithblackandredmatcheswithred. 9. RunconstantAmps:300mA(0.3A)for60,stirring. 10. Pourdown1XTransferBufferintohazardouswastebeakerinthehood.(DonotpourTransfer Bufferdownthesinkbecauseitcontainsmethanol). 11. Usemembraneforimmunoblotting. 12. Gelcanbesavedin1XElectrophoresisbufferforcoomasiestaining(~60 )anddestaining. 13. Prepare10mLofTBST(1xTBS+0.1%Tween)with5%milk: Volume Material 0.5g 10µL Drypowdermilk Bringto10mLwith1XTBS Tween 4
5 SelimeAksit/LIULAB LastRevised:6/22/10 Protocol Original6/8/ Blockforatleast60,gentleshaking,roomtemperature. 15. Prepare45mLofTBSTandwash3x Prepare10mLofTBST+5%milk+5 10µLofantibody. 17. Incubatefor60 gentlyshaking,rt. 18. Prepare45mLofTBSTandwashwith15mLincrements3x Ripapieceofplasticwrap.Pipettethe1mLofDURAWEST(500µLofbrownbottle+500µLofwhite bottle)ontothemiddleoftheplasticwrap. 20. Placethemembrane(sidewithproteins)ontothe1mLofDURAWEST.Useasmalltubetorollover thebacksideofthemembranetoremoveairbubblesanddispersethedurawestalloverthe membrane. 21. Waitfor5.RemoveexcessDURAWESTbyblottingwithkimwipe. 22. Ripanewpieceofplasticwrap,placethemembranerightsideup.Closeupthesidesoftheplastic wraptightlytoavoidthemembranefromdryingout. 23. EXPOSE! 5
Proper!initial!freezing,!subsequent!handling!and!sectioning!of!tissue!samples!will! eliminate!bad!freezing!artifacts.!!
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