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) 425px 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 500px gels
24mA per gel constant, 15°C, 6 -7 hrs