Hi Peter, You first want to reduce the protein concentration to avoid rapid precipitation upon adding crystallization solutions. Try concentrations of 5 - 10 mg/mL instead of 15 mg/mL. Next, consider changing the NaCl concentration. Salt can act as a co-precipitant, and lowering its concentration may increase solubility. Conversely, some proteins precipitate badly when salt is dialyzed out, so increasing the salt may also help. You also may consider using a different pH or different buffer (HEPES instead of Tris for example).
Glycerol is supposed to make proteins more soluble, but proteins that I have worked with have turned yellow when concentrated in the presence of 10% glycerol. Try dialyzing out the glycerol or using a buffer that does not contain any glycerol and see if you still see a yellow deposit on the concentrator or not. I think that the yellow color is caused by metal impurities in the glycerol, so using a higher purity glycerol may help too. If none of these suggestions help, you may want to consider using a fusion tag (maltose binding or SUMO) tag for one of your proteins. These tags can help increase protein solubility, but unfortunately may also disrupt protein-protein interactions. Matt
