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Re: [ccp4bb] rescuing crashing-out protein eluted from Nickel column |
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CCP4bb navigationCCP4bb <-- 2008 <-- February 2008 <-- 15 February 2008Subject: Re: rescuing crashing-out protein eluted from Nickel column From: "R {- dot -} M {- dot -} Garavito" garavito {- at -} MSU {- dot -} EDU Date: 2008-02-15 This is not a uncommon problem with Ni-chelation chromatography (on Ni-NTA or Talon) with soluble and membrane proteins. In most of my experiences, it is a salt problem (i.e., too little), but hydrophobicity issues abound, as well. On a pilot scale, just add any of the suggested additives (EDTA, DTT, extra salt, or even just a different buffer) to your tubes before taking fractions. Thus, the eluted protein goes drops into a "better" solution environment. If you set it up right, the protein is not exposed to high concentrations of any of the above, nor is diluted too much. For example, for 1 mL fractions, add 0.25-0.5 mL of buffer with the additives so the final volume per fraction is 1.25-1.5 mL. Other things to add/change are: (1) pH, (2) a little detergent or 0.5 M LiCl (to reduce hydrophobic interactions), (3) some NDSB compounds (non-detergent sulfobetaines; see the Anatrace or Sigma catalogs) or L-arginine to reduce non-specific aggregation, (4) and 0.25-0.5 M trimethylaminoxide. And the list goes on. It is tedious work, but not too difficult to do. Good luck, Michael **************************************************************** R. Michael Garavito, Ph.D. Professor of Biochemistry & Molecular Biology 513 Biochemistry Bldg. Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334 Email: garavito@msu.edu **************************************************************** On Feb 14, 2008, at 7:26 PM, Jacob Wong wrote: > Dear all, > > I just ran into this problem and would like to see if I could get > some helpful tips before my protein completely crashes out. > > I have a protein as 6His fusion and it remained bound to the Ni > resin with 40 mM Imidazole wash (added to 1XPBS) but then was > eluted off with 200 mM (added to 1XPBS). The protein seemed to be > highly concentrated in the elution and began to get cloudy right > away, with more and more precipitation produced over a matter of > minutes. I felt so helpless, didn't know what to do, and then > decided to add 5% of glycerol into one of the fractions but that > made it even more cloudy (ohh no...). > > While the protein is dying in the tube, do you have some quick > remedy for me? Thanks very much, -J.J. CCP4bb navigationCCP4bb <-- 2008 <-- February 2008 <-- 15 February 2008 |
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