Protein precipitated after buffer exchange
Posted 30 January 2005 - 11:46 AM
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!
Posted 02 February 2005 - 12:18 AM
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.