The protein is a small soluble protein around 14 Kda and is precipitating copiously beyond the deubiquitinylation step, especially on spin concentration. It is used in the pHUE vector system. When the ubiquitinase is added to the protein both are precipitating out.
Hi Bratati,
First, it might sound obvious, but remember that all molecules have a solubility limit, so check that you stay within a reasonable concentration range when you concentrate your sample.
The ubiquitin-less protein is probably not as soluble as the original fusion, and many proteins are soluble in a narrow range of conditions, so you might want to try several buffers (with various pH and salt concentrations) for solubilization. Also, depending on the protein, the nature of the buffering agents (PBS, TBS...) can influence protein solubility. Adding small solutes in the solution (glucose, glycerol, malonate...) might also help.
Do you need to preserve the secondary structure of your protein, for subsequent in vitro activity tests?
If so, producing your protein as a fusion with a small solubility-enhancing tag (e.g. the poly-ionic FLAG tag) may help you, without significantly modifying the protein activity.
If you have reasons to suspect that your protein can form disulfide bonds, use a bit of DTT to reverse intermolecular reactions that could drive aggregation.
Mild detergents and chaotropic agents can be used at low concentrations to disrupt intermolecular interactions while avoiding protein denaturation. Don't use too much, though.
Conversely, if you do not need to preserve the structure (e.g. for an electrophoresis or a quantification), you can use harsh conditions that might denature your protein but will easily solubilize it; for example you can heat your sample (around 95-100°C for a few minutes) in a buffer containing SDS.
I hope that helps!
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