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Urea Lysis Protocol

Urea Lysis Protocol

 

Urea lysis buffer

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

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

 

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 21C 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 -70C 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 -70C

 

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, 15C, 6 -7 hrs