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
