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 On Fri, Mar 20, 2009 at 9:32 AM, Tim Gruene <t...@shelx.uni-ac.gwdg.de> wrote: > 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 <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 >> >>
