Protein precipitated after buffer exchange - (Jan/30/2005 )
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!
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....
The problem doesn't arise from the pH, but from the retrieval of salts. Your buffer B doesn't contain any salt, and what is worse, it contains imidazole. Imidazole is known to cause precipitation and you definately need to remove it. Also, 0.5M imidazole is a bit high and will tend to precipitate the protein because it removes the hydration shell on the protein. My advice to store proteins at -20C is this: POTASSIUM PHOSPHATE buffer, and not sodium phosphate. In fact, NaPO4 buffers precipitate at low temperatures (-20C), thus unbuffering the solution and reducing the pH, which causes precipitation. But, with KPO4 buffers, the pH remains unchanged, hence preserving the solubility of the protein. I recommend reading on that subject (the author is John F. Carpenter) they did impressive work on protein stability upon freeze/thawing.
Also, in my experience with a his-tagged protein, KPO4 buffer resolved indeed my freeze/thaw problems. I use 150mM KCl, 75mM KH2PO4, pH 7-8.
One more problem I encountered (and haven't resolved yet) is the buffer exchanging on Amicon Ultra-15 columns. One othe factor that will cause aggregation of protein is dilution, which acts by denaturing proteins. You can try this simple experiment: take a concentrated protein, and dilute it in the same buffer, say 10 times. You will noticed the aggregation. If this is the case, then you should avoid buffer exchanging by dilution/reconcentration. I'm currently working on this problem, and i'll keep you updated when I find a solution. Good luck.
Do you think oxygen should be eliminated in its entirety? Has anyone done double distilled water and autoclaved and sealed to prepare ALL THEIR SOLUTIONS AND BUFFERS?