precipitatio of protein - not during purif but in concentration... (Sep/15/2006 )

i'm currently doing a protein concentration.
The purification on column with FPLC goes well. I do a His column fowllowed by monoQ and then gel filtraton. At final step i get my protein in a soluble form in buffer : Tris 50mM pH 8.5 NaCl 150mM DTT 1mM EDTA 0.5mM and CHAPS 0,1mM, at 0.53 mg/ml.

For my purpose i need to concentrate it. For that, i'm using an amicon column with a cut off of 10kD (my protein is 25kD). The concentration works well until i reach the concentration of 5.53mg/ml.

I don't understand what goes on...
What do you think i would try for that? Is it a critical concentration after which my protein would precipitate?....

you could add some reagent(s) after final column elution to increase solubility at that concentration?

Fred, I assume that you used table top centrifuge to concentrate it? Any chance that the temperature was not controlled well and got too warm during the concentration process? Also check to see if adding TW-80 0.1%, reducing DTT to 0.1 mM, plus 10% glycerol would help with reducing your precipitation problem.

i've worked with proteins that would aggregate if you concentrated them over a certain concentration when they were pure. i found that adding peg-8000 up to a maximum of ~5% allowed me to extend the concentration where it would aggregate.

if you try peg then be careful, peg-8000 will concentrate on amicon with 10kDa cutoff so start with lower concentration or you may precipitate your protein with the peg (i make my protein in buffer containing 0.05% peg and concentrate ~20x thereby giving ~1% peg).

ok thanks for the replies
As i like to understand well what goes on in my tubes, may you tell me what is the aim of reducing DTT concentration ? The process is not done in a table top centrifuge.
i'm currently away from the lab but will try your recommendations.

genehunter : do you think that using tween 80 instead of CHAPS will do sthg relevant? I don't really know the differences of these detergents, but need to check.
Thanks again.

PS : i wanted to add urea to a 4M final concentration, but as it's for cristallization of the protein i'm not sure of the possibility to recover the protein if i dyalize after concentration. What do you think about that?

I am just worried about the possibility of DTT break S-S and cause intermolecule cross-linking. This would be more likely to happen when protein concentration is high. Maybe compltely removal of it is an extreame condition to consider? TW-80 is just an alternative to CHAPS and I dont have a strong reason for it. But it does provide certain shielding effect between proteins, because its larger size tha CHAPS. Is it a membrane protein, by the way?