Yes!
And if you have money, you can use phosphoramidon
http://delphi.phys.univ-tours.fr/Prolysis/phosphoramidon.html
It's nearly as good as EDTA or phenantroline for inhibiting metalloproteases
- and it's fully compatible with IMAC!
Artem
-----Original Message-----
From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of
[log in to unmask]
Sent: Sunday, November 04, 2007 5:18 PM
To: [log in to unmask]
Subject: Re: [ccp4bb] protein degradation?
Some proteases are metal-dependent, and inhibitors for those aren't
Ni-column-compatible. "We" (meaning my students) found that it helps
to (1) work very quickly and (2) put EDTA into the fraction collector
tubes before eluting from the Ni column.
At 06:22 AM 11/4/2007, Vijay Kumar wrote:
>Hi,
>
>I have been trying purify a N-ter his-tagged protein over-expressed
>in E.coli. After purification (Ni-NTA or Co-Talon), I find two bands
>in SDS PAGE which are very close each other (top band in the right
>MW and more intense than the lower band). Western blot (for his-tag)
>of the gel gave signal for both the bands. Mass spec results
>confirmed both protein bands are the same. So I think it could be
>C-ter degradation of my protein. Also the 2 bands exist after
>ion-exchange and sizing column.
>
>I use commercially available complete protease inhibitor tablets
>(increasing concentration has no effect) and sonication for lysis. I
>am wondering if people have encountered the same problem and got any
>suggestions?
>
>
>Thanks in advance.
>
>Regards,
>
>Vijay
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Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
fax 773 702 0439
http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alpha
betically.php?faculty_id=123
http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html
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