Urea Lysis Buffer

Hi,
I looked for a Urea Lysis buffer,
I found this one on line and it works:

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

The protocol was taken from this website:
http://www.scripps.edu/mem/memcore/proteomics/Protocols4.htm

I used my regular protocol to run the samples for IEF.

Dear Gsovak, May I know the function of urea at high concentration in your study? Recently, I'm doing an extraction based on someone's method. It says that I have to add in 10 M of Urea before the extraction started. I'm just curious why we need urea at high concentration. TQ

Hi Mie: Urea is used for denaturing proteins because it is a chaotrope at high concentrations and denatured proteins can be readily solubilized on lysis buffer. Also urea helps in cysteine blocking reaction with agents such as iodoacetamide or acrylamide. Sometimes urea can react with cysteines to form carboxymethylcysteines especially when is heated over 60oC.