Dear Kien,
you might also try a different resin/ metal ion. If I remember correctly,
the technician where I did my PhD had much better results with a Talon
resin using Co instead of Ni: you can use much less Imidazole, only 5-10mM
for washing and 50mM for elution.
If you can go back to cloning, try a C-terminal fusion protein. That
should prevent you from purifying shorter product caused by truncation of
translation: if the His-tag is at the C-terminus, everything before (your
target protein) would be there, too!
Tim
--
Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen
GPG Key ID = A46BEE1A
On Fri, 20 Mar 2009, Phoebe Rice wrote:
Try running the Ni column as fast as possible and putting
concentrated EDTA in the fraction collector tubes before you
start, to minimize opportunities for metal-dependent proteases.
It may not be a magic bullet but it can't hurt.
Phoebe
---- Original message ----
Date: Thu, 19 Mar 2009 23:53:14 +0000
From: Kn Ly <kn...@auckland.ac.nz>
Subject: [ccp4bb] purification
To: CCP4BB@JISCMAIL.AC.UK
Hello everyone,
I am expressing a 100 KDa eukaryotic membrane protein in E
coli. The protein
is fused to 6His-MBP in the N terminus and the resulting mass
is ~ 150 KDa.
However, the protein get severely degraded so after putting
through a Ni-NTA
column, the protein came out with a lot of contaminant bands.
I did a
western blot using antibody against his tag. The total cell
lysate gave
signals in many bands. The flow through did not give any
signal and the
eluted fraction again gave many band signals, indicating the
protein got
degraded copiously even before purification.
I used Roche protease inhibitor tablet and still got a lot of
degradation.
Can anyone suggest a way to avoid the problem or a
purification method so
that I can purify the intact protein while keeping away the
unwanted
degraded fractions.
Thanks heaps in advance.
Kien
Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123
RNA is really nifty
DNA is over fifty
We have put them
both in one book
Please do take a
really good look
http://www.rsc.org/shop/books/2008/9780854042722.asp
