It's possible that the protein you are investigating normally exists as part of a high molecular weight complex or oligomer, rather than being aggregated. High salt treatment may disrupt this complex and allow the protein to be isolated by itself, but beware of the chaotropic (denaturing) effect of high NaCl concentrations. You may want to try a variety of salt concentrations to find the minimal effective concentration for eluting your protein after the void volume.

If you use detergent, you have to consider that proteins incorporated into micelles will elute at the molecular weight of the protein + micelle. In other words, the proteins will appear larger than they are. Some detergents have very small micelles (example: CHAPS) that minimize this problem. Also, make sure to use enough detergent that each protein molecule can be in its own micelle. Otherwise, proteins may coelute because they share a micelle rather than because they form a complex with each other.

Hi there,

If it's actually an issue with aggregation then it would be better to try to prevent it rather than to cure it. The easiest is to start your extraction/purification process with high salt concentration (0.5M-1M NaCl) and to keep it high in all the buffers used prior and during SEC. And see what happens. The use of detergent is a bit more tricky as it might also solubilize unfolded/denatured proteins

Thanks for the suggestions.

Currently I am running SEC with 0.1% Tween-20 in the sample and elution buffer so we'll see what that does.

I have been taking 500 mL of protein secreted into the fungal media and concentrating it to ~50 mL - I imagine this might where the aggregation is occurring.  Next time I purify some protein I will try to buffer exchange the supernatant into high NaCl (1 M) before concentration in an attempt to reduce aggregates from forming.

You don't have to do a buffer exchange. You can just add solid NaCl, or use a 5M stock solution.

Have you optimized the pH? What is the pI of your protein? You could try optimizing pH and salt both, or even try a hint of glycerol. What purification steps are you using prior to SEC?

The problem is I don't know what the protein is.  I am trying to isolate it based on activity because ultimately I would like to express it recombinantly.  SEC is the first step of purification after concentrating the supernatant.

I am also interested in this. My protein has aggregated after SEC. Buffer it was in before had 1% Tween, elution buffer however did not, so I suspect it might re-solublize in the original buffer. Otherwise am not sure what to do in order to proceed.

Any suggestions?? How did the salt go for you?

When I repeated the protein concentration from the media I included 0.1% Tween-20.  In the subsequent SEC the aggregate peak was significantly reduced and the activity I observed was much improved over previous runs probably because my target proteins wasn't in the aggregate any more.

Patrick - I would suggest including whatever level of Tween you have in the protein before SEC in the buffer used for the SEC, although 1% is higher than I used.  0.1% for me was sufficient to make a big difference.  I also tried 0.5 M NaCl instead of Tween but it didn't seem to help.  My best efforts to date are using 0.1% Tween-20 in the the supernatant mixture before protein concentration, and using 100 mM NaCl and 0.1% Tween-20 in the SEC elution buffer.  I hope that helps.