Hi all,
I have a protein that needs to be purified with 0.05% Tween-20 in the buffer. My question is, can I concentrate this protein afterward with a centrifugal concentrator (10kDa)? I think 0.05% Tween-20 is already above its CMC (based on this link, https://openwetware.org/wiki/Critical_micelle_concentration_(CMC)), will micelles formed in the buffer get stuck in the concentrator and not pass through, and increase my Tween-20 concentration in the protein sample? Are there any ways I can determine the Tween-20 concentration in my sample?
Good question. So the cmc of Tween 20 is reported to be 60 mg/L (note, I only looked at one reference, so please check on this). And 0.05% Tween 20 would be a concentration of 500 mg/L. So yes, the "total" concentration is about 10 times the cmc. However, in you protein solution, most of the tween will be adsorbed to the protein, so the question really is, how do you remove the tween from the protein. (to answer the question you asked, no, you won't likely get micelles if you concentrate, as you likely have only monomers in solution (and 90% of the tween adsorbed on the protein) and the monomers will pass through the 10kDa membrane, leaving the remaining tween still associated with the protein.) - What I think you need to consider is how can you enhance the removal of the tween from the protein. You may want to test a few solvents to see if you can solubilize more of the tween in solution before concentrating. It may take several "washing" steps to remove the surfactant. Maybe try 5% ethanol, or other water miscible solvents. Essentially you would be replacing the surfactant (on the protein surface) with a water miscible solvent that can be more easily removed by followup washings with pure water. Just some thoughts.
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