Urea lysis buffer

            9M Urea, 2.5mM EDTA, 2.5mM EGTA, 1% DTE, 4% CHAPS

            make 10ml and aliquot 10x1ml, freeze at -70°C

Lysate preparation

            wash the cells 2x with PBS

            wash the cells 1x with 10mM Tris, 250mM Sucrose

            lyse the cells with 100 – 350ul of urea lysis buffer (depending on # of cells and strip size)

            lyse at room temperature for 30 – 45 min, vortexing every 10 min

            transfer lysate to ultracentrifuge tubes and spin at 50000 RPM at 21°C for 90 min

            apply the supernant to a Qiagen QIAshredder (cat#79654), spin at 14000 RPM for 2 min

            save 20ul for Protein Assay

            freeze sample at -70°C to run 1D later or continue on

Sample application during rehydration

            + bromophenol blue + ampholytes to samples

            in a rehydration tray + samples, lay strips face down in sample

+ mineral oil, incubate 15 – 18 hrs

IEF (1D) 17cm pH 4-7 BioRad

            Step 1  250V               1 hr                  linear

            Step 2 10000V            2 hrs                linear

            Step 3 10000V            45000 VH        rapid

            Place strips face up in equilibration tray and freeze in -70°C

Equilibrate strips

            1 x 10min 375mM Tris-HCl pH8.8, 6M Urea, 2% Urea, 2% DTT, 30% glycerol

1 x 10min 375mM Tris-HCl pH8.8, 6M Urea, 2% Urea, 2.5% Iodoacetamide, 30% glycerol

wash strips with gel running buffer

SDS-PAGE

            Run strips on 12% Acrylamide 18 x 20cm gels

            24mA per gel constant, 15°C, 6 -7 hrs