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(revision1/05/2001)

SDSPageGelElectrophoresis
PAGE
Polyacrylamidegelelectrophoresis(PAGE)isprobablythe
mostcommonanalyticaltechniqueusedtoseparateand
characterizeproteins.Asolutionofacrylamideand
bisacrylamideispolymerized.Acrylamidealoneforms
linearpolymers.Thebisacrylamideintroducescrosslinks
betweenpolyacrylamidechains.The'poresize'is
determinedbytheratioofacrylamidetobisacrylamide,
andbytheconcentrationofacrylamide.Ahighratioof
bisacrylamidetoacrylamideandahighacrylamide
concentrationcauselowelectrophoreticmobility.
Polymerizationofacrylamideandbisacrylamidemonomers
isinducedbyammoniumpersulfate(APS),which
spontaneouslydecomposestoformfreeradicals.TEMED,a
freeradicalstabilizer,isgenerallyincludedtopromote
polymerization.
SDSPAGE
Sodiumdodecylsulfate(SDS)isanamphipathicdetergent.
Ithasananionicheadgroupandalipophilictail.It
bindsnoncovalentlytoproteins,withastoichiometryof
aroundoneSDSmoleculepertwoaminoacids.SDScauses
proteinstodenatureanddissassociatefromeachother
(excludingcovalentcrosslinking).Italsoconfers
negativecharge.InthepresenceofSDS,theintrinsic
chargeofaproteinismasked.DuringSDSPAGE,all
proteinsmigratetowardtheanode(thepositivelycharged
electrode).SDStreatedproteinshaveverysimilar
chargetomassratios,andsimilarshapes.DuringPAGE,
therateofmigrationofSDStreatedproteinsis
effectivelydeterminedbymolecularweight.

Belowisanexampleoftheprocedureforperforming
discontinuousSDSPAGEwitha14%separatinggelanda5%
stackinggel.
Materials

PAGERigsincludingglassplates(10x20cm),spacers,
comb,andclamps
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Proteinsample
BioRadLaemmliSampleBuffer(containsSDSandeither
sucroseorglycerol)
2Mercaptoethanol(reducesdisulfidebonds,disrupts
proteincrosslinks)
MWMarkers(alreadypreparedinsamplebuffer)

GelCassetteAssembly(BioRadMiniProtean3)
Cleanandcompletelydrytheglassplates,combs,and
anyotherpertinentmaterials.
Placeashortplateontopofaspacerplate.Insert
bothplatesintothegreencastingframeonaflat
surface.Besurethatthe"legs"ofthecastingframe
aredown.Clampthecastingframeandcheckthatthe
platesarelevelonthebottom.
Putthecastingframeintothecastingstand.
PreparationoftheGel
CombineallreagentsexcepttheTEMEDforthe14%
separatinggel.
14%SeparatingGelComponents(4.195mL)
1.184millilitersdeionizedwater

1.96milliliters30%acrylamide/Bis(Warning:
Acrylamideisaneurotoxin.Usegloves,donot
ingest.)
1.0milliliters1.5MTris,pH8.8
21microliters20%SDS
12microliters10%ammoniumpersulfate
18microlitersTEMED,pH8.9
Whenreadytopourthegel,quicklyaddtheTEMED,
mixusingaPasteurpipette,andtransferthe
separatinggelsolutionbetweentheglassplatesin
thecastingchambertoabout3/4inchbelowtheshort
plate.
Asmalllayerofbutanolcanbeaddedontopofthe
gelpriortopolymerizationtostraightenthelevel
ofthegelandremoveunwantedairbubblesthatmay
bepresent.Butanolwillnotmixwiththeaqueous
separatinggelsolution.Oncethegelhas
polymerized,thebutanolcanberemovedbyabsorption
withKimwipesorfilterpaper.Rinsethetoplayerof
thegelwithdIwaterpriortopouringthestacking
gel.
Insertthewellformingcombintotheopeningbetween
theglassplates.
CombineallreagentsexcepttheTEMEDforthe5%
stackinggel.
5.1%StackingGel(2.484mL)
0.4millilitersdeionizedwater
1.8milliliters30%acrylamide/Bis
0.25milliliters0.5MTris,pH6.8

12microliters20%SDS
17microliters10%ammoniumpersulfate
5microlitersTEMED
Whenreadytopourthegel,quicklyaddtheTEMED,
mixusingaPasteurpipette,andtransferthe
stackinggelsolutionbetweentheglassplatesinthe
castingchamber.
Boththeseparatingandstackinggelsshould
polymerizewithinsixminutes.
Oncethestackinggelhaspolymerized,thecombcan
begentlyremoved.Thepolymerizedgelbetweenthe
shortplateandspacerplateformsthe"gel
cassette".
SamplePreparation
Placesomewaterina600mLbeakerandleaveona
hotplatetoboil.(Thiscantake15minutesor
more.)
Meanwhile,add50mLof2mercaptoethanolto950mL
ofLaemmlisamplebuffer.
Combine10mLofeachproteinsamplewith20mLof
Laemmlisamplebufferplus2mercaptoethanolin
microcentrifugetubes.
Inseparatetubes,aliquot10mLofMWmarker.(MW
markersarealreadypreparedinLaemmlisample
buffer.)
Boilthesamplesfornomorethan5minutestofully
denaturetheproteins.Leavethesamplesatroom
temperatureuntilreadytoloadontothegel.

Electrophoresis
Removethegelcassettefromthecastingstandand
placeitintheelectrodeassemblywiththeshort
plateontheinside.
Slidetheelectrodeassembly(withthegelcassette)
intotheclampingframe.Pressdownontheelectrode
assemblywhileclampingtheframetosecurethe
electrodeassembly.Thisstepisimportantto
minimizepotentialleakageduringtheelectrophoresis
experiment.
Poursome1Xelectrophoresisbufferintotheopening
ofthecastingframebetweenthegelcassettes.Add
enoughbuffertofillthewellsofthegel.Useagel
loadingtiptopipettesomebufferintoeachwellto
ensurecleanliness.
Whenallwellsaresufficientlycleaned,slowly
pipettenomorethan30mLofdenaturedsampleorMW
markerintoeachwell.Ayellowguidecanbeplaced
ontopoftheelectrodeassemblytoaidinloading
thegel.
Whenthegelhasbeenloaded,lowertheclamping
frameintotheelectrophoresistank.
Filltheregionoutsideoftheframewith1X
electrophoresisbuffer.
Coverthetankwiththelidaligningtheelectrodes
(blackorred)appropriately.
Connecttheelectrophoresistanktothepowersupply.
Allowthesamplestorunat30mAuntilthedyefront
reachesthebottomofthegel.Thiscantakeaslong
as1hour.

 Whenelectrophoresisiscomplete,turnoffthepower
supplyanddisassembletheapparatus.