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Urea Lysis Buffer

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gsovak

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Joined: Jan 25, 2005







 Send a personal messsage to gsovak Reply with a quote from this post Go to the top of the page

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.

.........................

Posted Jun 14, 2006, 12:44 PM
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