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:[EMAIL PROTECTED] On Behalf Of [EMAIL PROTECTED] Sent: Sunday, November 04, 2007 5:18 PM To: [email protected] 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 ---------------------------------------------------------------------------- ----------------------------------------------- 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
