CastingandrunningproteingelsaccordingtoLaemmliusingBioRad’sMinipro-teanII.
Theprotocol
1.Taketwospacers,acomb,onesmallandonelargeglassplate,acastingblockandthecastingstand.2.Cleantheglassplateswithethanol.
3.Assemblethe“sandwich”,takecasethattheloweredgesofblassplatesandspacersarewellaligned.Otherwisethewholethingwillleakwhenyoupourthegel.4.Prepare10%(w/v)ammoniumpersulfate(APS)solutionfresh
5.Preparetheseparatinggelmixture(atleast10mlpergelwhenusingthe1.5mmspacers):
30%acrylamide
10%gel12%gel15%gel18%gel
BufferI2.5ml2.5ml2.5ml2.5ml
MilliQ4.0ml3.3ml2.3ml1.3ml
3.3ml4.0ml5.0ml6.0ml
6.Add100lof10%SDS,80lof10%APSand10lofTEMED
7.Pourthegelleavingca.2cmfreefromthetopofthelowerglassplate.Care-fullypipettewaterorwater-satyratediso-butanolontoptocreatesmoothtopsurface.8.Whentheseparatinggelhaspolymerised,preparethestackinggelmixture(3.75%acrylamide):
2.4ml1.0ml0.5ml40l
MilliQBufferII
30%acrylamide10%SDS
9.Pourthewaterfromthetopoftheseparatinggelcompletely.
10.AddAPSandTEMEDtostackinggelmix,pourittothetopandinsertthe
comb.Becarefulnottointroduceairbubblestothegel.Letitpolymerise.11.Attachthegelsandwichtotheelectrodeassembly.Greasethegaskettopre-ventleakage.Ifyouarerunningonlyonegel,turnthegasketfromtheothersidearoundandputonlytheacrylblockonthatside.12.Pourrunningbufferinsidethegelassembly.Thebuffershouldcoverbein
contactwiththegel.Letstandforfewminuteswhilepreparingthesamplestocheckforleakage.Ifthereisnoleakage,filltheouterchamber(tank)withbuffertocoverthebottomofthegels.
13.Preparethesamplesbymixingonevolumeofsampleandonevolumeof
samplebuffer.Heatat95Cforfewminutesandloadonthegel.
14.runwith150Vforca.1h20min.
Solutionsneeded
BufferI(4x)BufferII(4x)30%AcrylamideSamplebuffer(2x)
1.5MTris-HClpH8.80.5MTris-HClpH6.829.2%acrylamide0.8%bis-acrylamide
0.125MTris-HClpH6.820%glycerol4%SDS
2%-mercaptoethanol(-ME)0.02%bromphenolblue
Runningbuffer(1x)
25mMTris
192mMglycine0.1%SDS
Notes
Acrylamideisverytoxicandshouldbehandledwithcare.Wearalwaysglovesandcleananyspillsetc.immediately.Ifyouhavetoweightitout,doitinthefumehudeasthepowderisverylight.Iwouldrecommendtobuyreadymadesolutionswheneverpossibletoavoidtheneedtohandlethepowder.
Preparethesamplebufferwithout-MEandadditafterthawingnewbatchfrom-20C.Fornon-reducedsamplesleaveitout.Inpracticetomake100mlmixallthecomponentsexcept-MEandfillthevolumeto98ml.Freezethesolutionin9.8mlaliquotes.Add200lof-MEbeforeuse.
Runningbuffercanbepreparedas5-or10-foldstocksolution.Tomake5lof5-foldstock,youwillneed75.6gofTrisbase,360gofglycineand25gofSDS.DonotadjustthepH,itshouldbecorrectasis.
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