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 <[log in to unmask]>
>> Subject: [ccp4bb] purification
>> To: [log in to unmask]
>>
>> 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
>
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