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
