Dear Hard Hard

Just some questions:

1) Which lysis buffer did you use?

2) Is you protein contain transmembrane regions

3) Did your protein contain Cysteines or metal binding motif?

If yie, 3a) Did you run the protein precipitate in SDS page in reducing and non reducing condition?

The precipitation after thawing at -20°C is something quite common also with stable and well folded protein in my experience and it can be prevented by adding some glycerol (20%) in the protein solution before formulation.

Slow precipitation at 4°C, after IMAC purification is something less frequent that in my experience is due to an intrinsic instability of the protein, and that aggregation propensity was due to:

1) presence of some hydrophobic region (eg an uncutted signalp peptide, or transmembrane elix)

2) Presence of metal binding site, that may strip some metal from the

3) presence of free cysteines that in absence of reducing agent tend to form unspecific S-S bonds between the different molecules and lead the aggregation:

in my experience, you can prevent in the case 2 just by adding some EDTA (eg 10mM) to the solution just after protein elution, in the case 3 by adding reducing agent in the elution but if the Cysteines are involved in structural S-S bond formation in this way you will obtain an unfolded protein and in case you would like to produce it in folded conformation you need to change your production approach (eg use a different expression strain or hosts)

in case 1) the solution is more difficult. You have to design a different protein domain.

as general comment, i suggest to you to replace dialysis with desalting with is more fast.

I do not know if one of this solution could be good for you, but you can check at least the case 2 and 3 in short time.

good luck

Manuele

Thank you so much for your detailed answer. I learned a lot from your shared experience. I think I will first try adding EDTA after eluting. My protein is a secreted protein from bacteria, it is not a transmembrane protein and has no Cys in its sequence. The lysis buffer I used is PBS with lysozyme, RNase, DNase, MgCl2, PMSF. Thank you again for your lesson.

You should not store proteins in phosphate buffer at -20oC, I have been told, because this buffer undergoes a phase transition in that temperature range that can destabilize proteins. You could flash-freeze the protein and store is at -80oC, or you could store it at -20oC in the presence of 50% glycerol to keep it from freezing, or you could use a different storage buffer.

I generally avoid storing proteins frozen at -20oC anyway, because over a period of a few months, the ice sublimes from the surface, leaving the protein dry. This can be prevented by storing the protein at -80oC.

Do not allow protein solutions to freeze slowly. They should be flash-frozen to prevent separation of solutes out of the ice.

Thank you! I will keep in mind your advice of protein storage. That is an important step after all. I should follow to preserve the protein's quality.