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Re: [ccp4bb] crashing-out protein eluted from Nickel column |
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CCP4bb navigationCCP4bb <-- 2008 <-- February 2008 <-- 19 February 2008Subject: Re: crashing-out protein eluted from Nickel column From: price {- at -} UCHICAGO {- dot -} EDU price {- at -} UCHICAGO {- dot -} EDU Date: 2008-02-19 of our wimpier proteins, spiking each fraction collector tube with a little EDTA before running the Ni column really helped reduce keep the sample in one piece. Phoebe At 01:18 PM 2/19/2008, Sophia Tsai wrote: >Hi, > >Agreed on this. I used to have issues with aggregation due to the >Nickel being stripped off the column (happens with elution in >imidazole). Adding 10mM EDTA to the elution immediately AFTER it has >come out of the column will chelate the Ni++ and prevents >aggregation (at least in my case). Afterwards, I use gel filtration >to remove the Ni++, as well. > >Hope that helps! >Sophia > >On Mon, Feb 18, 2008 at 4:48 AM, Ngo Duc Tri >< >Hi, >I used another way to deal with this problem. You can try to elute >your protein with the buffer containing 50mM EDTA (You need at least >10CV to elute completely). Then use gel filtration to remove the Ni. > >I applied this method with two proteins and it showed good results. >Good luck! > >TriNgo > --------------------------------------------------------------------------------------------------------------------------- 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_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 CCP4bb navigationCCP4bb <-- 2008 <-- February 2008 <-- 19 February 2008 |
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