Hi Kien,
As Artem pointed out earlier. Are you sure that your protein folded correctly. You want to make sure that the protein is expressed in lipid membrane not in inclusion body. If so, considering changing the host might be a good idea.
Also, did you use any kind of detergent when you extract the protein? Choice of detergent is also very important in purifying a membrane protein. Try different detergents if you havent used one and it may work.
Talon resin works fine with me when I was working with ion channel.
Hope that helps,
Puey
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 +0000coli. The protein
From: Kn Ly <kn.ly@AUCKLAND.AC.NZ>
Subject: [ccp4bb] purification
To: [log in to unmask]
Hello everyone,
I am expressing a 100 KDa eukaryotic membrane protein in E
is fused to 6His-MBP in the N terminus and the resulting massis ~ 150 KDa.
through a Ni-NTA
However, the protein get severely degraded so after putting
column, the protein came out with a lot of contaminant bands.I did a
western blot using antibody against his tag. The total celllysate gave
signals in many bands. The flow through did not give anysignal and the
eluted fraction again gave many band signals, indicating theprotein got
degraded copiously even before purification.degradation.
I used Roche protease inhibitor tablet and still got a lot of
Can anyone suggest a way to avoid the problem or apurification method so
that I can purify the intact protein while keeping away theunwanted
degraded fractions.Phoebe A. Rice
Thanks heaps in advance.
Kien
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