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Concentrating Protein - protein precipitates upon concentrating (Jun/04/2009 )

I have been trying to concentrate protein for crystallization. Several papers have concentrated the same protein to 20 mg/mL and they do not specify their concentrating methods with luxury of detail (since it may be a trivial topic to them). I have achieved 5 mg/mL concentrate at which point it precipitates; my protein has a 6-His-Tag (this is the only difference). I use the Amicon concentrator tubes and centrifuge for 5 minutes, stop and mix the sample, and repeat this process (while adding more diluted protein from purification at each step). The buffer I"m using is sodium phosphate pH 6.7. I have also used sodium acetate in an attempt to decrease the pH, to 5.3, since this might be a possible issue.




I imagine it's the his tag causing you the problem. Sometimes a his-tag can make the protein less stable in your case precipitate out in high conc.

I had this problem with a protein before and determined it was the tag.

I'd suggest going with the untagged if you can?