Urea Lysis Protocol

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

make 10ml and aliquot 10x1ml,freeze at -70℃

Lysate preparation

wash the cells 2x with PBS

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

lyse the cells with 100 – 350μl 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 μltracentrifuge tubes and spin at 50000 RPM at 21℃ for 90 min

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

save 20μl for Protein Assay

freeze sample at -70℃ 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℃

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℃,6 -7 hrs