Something else you could try is adding know ligands to your purification
step - depending on your proteein (or even during expression, e.g.
metals come to mind).
Juergen
[EMAIL PROTECTED] wrote:
Just beware that changing how you break the cells open can
change the average size of chromosome chunks, which can
change how DNA binding proteins behave in the lysate.
---- Original message ----
Date: Mon, 3 Mar 2008 15:21:15 +0000
From: Mads Gabrielsen <[EMAIL PROTECTED]>
Subject: [ccp4bb] finicky protein
To: CCP4BB@JISCMAIL.AC.UK
I am not a big fan of sonication. Try changing your way of
disrupting the
cells.
I have compared sonication vs mechanical stress on several
unrelated proteins,
and for me a good old french press wins every time. If you
want to get all
modern and fancy, a cell disruptor gives similar results.
Cheers,
Mads Gabrielsen
[Hide Quoted Text]
On Mar 2, 2008, at 11:47 PM, Tim Gruene wrote:
Hi all
sorry, for offtopic query...
I am trying to purify my protein by Ni-NTA affinity
chromatography. After
sonication as i centrifuge bacterial lysate, soon after 10
min whole
lysates
get precipitated during loading on the column and some time
it remain
soluble too. if i get purified through the column without
precipitation,
it
gets precipitated during dialysis.
I have tried lot, by chnaging buffers, increasing salt or
deacreasing salt
or no salt at are helpless.
I do purifiaction in cold room.
can any one suggest some solution?
Thanks in advance.
NSH
--
Dr. Mads Gabrielsen
GBRC, B217
Division of Biochemistry and Molecular Biology
IBLS
University of Glasgow Phone Office: 01413308119
G12 8QQ Phone Lab: 01413306449
UK E-mail:
[EMAIL PROTECTED]
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Jürgen Bosch
University of Washington
Dept. of Biochemistry, K-426
1705 NE Pacific Street
Seattle, WA 98195
Box 357742
Phone: +1-206-616-4510
FAX: +1-206-685-7002
Web: http://faculty.washington.edu/jbosch
