Urealysisbuffer

9MUrea,2.5mMEDTA,2.5mMEGTA,1%DTE,4%CHAPS

make10mlandaliquot10x1ml,freezeat-70°C

Lysatepreparation

washthecells2xwithPBS

washthecells1xwith10mMTris,250mMSucrose

lysethecellswith100–350ulofurealysisbuffer(dependingon#ofcellsandstripsize)

lyseatroomtemperaturefor30–45min,vortexingevery10min

transferlysatetoultracentrifugetubesandspinat50000RPMat21°Cfor90min

applythesupernanttoaQiagenQIAshredder(cat#79654),spinat14000RPMfor2min

save20ulforProteinAssay

freezesampleat-70°Ctorun1Dlaterorcontinueon

Sampleapplicationduringrehydration

+bromophenolblue+ampholytestosamples

inarehydrationtray+samples,laystripsfacedowninsample

+mineraloil,incubate15–18hrs

IEF(1D)17cmpH4-7BioRad

Step1250V1hrlinear

Step210000V2hrslinear

Step310000V45000VHrapid

Placestripsfaceupinequilibrationtrayandfreezein-70°C

Equilibratestrips

1x10min375mMTris-HClpH8.8,6MUrea,2%Urea,2%DTT,30%glycerol

1x10min375mMTris-HClpH8.8,6MUrea,2%Urea,2.5%Iodoacetamide,30%glycerol

washstripswithgelrunningbuffer

SDS-PAGE

Runstripson12%Acrylamide18x20cmgels

24mApergelconstant,15°C,6-7hrs

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