lysis buffers, zymography and EDTA

John Ladasky ladasky at my-deja.com
Sat Nov 9 01:40:37 EST 2002


Jen <jrobertson1 at sympatico.ca> wrote in message news:<40gesu0jmd6o6gl3qr1ja16cp5ri4js25q at 4ax.com>...
> I use a standard PLC lysis buffer containing EDTA. I'm making whole
> cell lysates for zymography. 
> 
> (1) I'm assuming that the EDTA would kill the enzymatic activity of
> the proteins I'm interested in (bad thing)
> (2) can I re-make the lysis buffer and just omit EDTA?

Hi, Jen,

There's another person in the newsgroups who asked a question about
lysis buffers today.  I answered her and you may be interested in my
reply.

http://groups.google.com/groups?dq=&hl=en&lr=&ie=UTF-8&oe=UTF-8&threadm=c09b237b.0211081511.dcbac59%40posting.google.com&prev=/groups%3Fhl%3Den%26lr%3D%26ie%3DUTF-8%26oe%3DUTF-8%26group%3Dbionet.molbio.methds-reagnts

I use an EDTA-free lysis buffer in my work, and I provide the recipe. 
What exactly is your "standard PLC lysis buffer"?

As far as I know, the main point of including EDTA in lysis buffer is
to inhibit divalent cation-dependent proteases.  Other protease
inhibitors may be able to substitute for EDTA.  Is your enzyme of
interest itself a protease?  That could get complicated.

Good luck!

--
John J. Ladasky Jr., Ph.D.
Department of Biology
Johns Hopkins University
Baltimore MD 21218
USA
Earth



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