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Protein precipitated after buffer exchange

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#1 netnus



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Posted 30 January 2005 - 11:46 AM

Dear friends,

I have been facing this trouble for a while: I want to exchange the buffer for my protein. I use Amicon Centriplus YM-3 with MWCO=3,000. My protein is soluble and stable in buffer A: KH2PO4 75mM,NaCl 75mM,4mM 2-Mercaptoethanol,2mM EDTA and 1mM DTT,5mM NaN3). The new Buffer B is:0.5M imidazole.HCl,NaEDTA 2mM and DTT(1mol/mol protein). After exchange for 4 times, the solution is concentrated down to 1ml. Then I store it in the -20 degree freezer. I thaw it and spin it down . My protein is almost completely precipitated.
Had anyone ever met with such a problem? How did you solve it?Any suggestion will be greatly appreciated!


#2 snolan6



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Posted 02 February 2005 - 12:18 AM

Have you made sure the pH of your buffer is permissive to your protein being soluble? Bad stuff like that happens to me sometimes when I try out a new protocol or make up a new buffer recipe, and pH is always my first candidate cause.

Also, make sure your pH electrode is functioning properly - ours has given me problems a few times.

Edit - the temp of your buffer can affect the pH also....

Edited by snolan6, 02 February 2005 - 12:28 AM.

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