Hello Everyone,

I have purified a protein called “protein-1” an innate immune protein from HEK293T cells, there is no crystal structure of this protein yet and I am able to purify milligrams of this protein from HEK293T cells. For now, I plan to get a small angle x-ray scattering (SAXS) structure of this protein and for that I need to concentrate it at least to 1 or 2 mg/mL. Unfortunately, I am not able to concentrate this protein beyond 0.6 to 0.7 mg/mL, it starts to precipitate as shown in the image and I loose most of my protein during this process which is very unfortunate considering it takes several days to purify it from mammalian cells as you all know. The molecular weight of the protein is 69KD.

Following are buffers that I have used so far and don’t work for concentration-

1. 50mM Tris PH 7.5, 100mM Nacl, 1mM DTT, 10% Glycerol

2. 50mM Tris PH 7.5, 50mM Kcl, 100mM Nacl, 5mM DTT, 20% Glycerol.

I am using Amicon ultra concentrators (10K or 15K) at 3500RPM to concentrate it at 4 degree Celsius. My supervisor has suggested me some new buffers that I will be trying this week. I would like to know your suggestions about different types of buffers, buffer concentrations and new methods to concentrate that could prevent protein from crashing in the solution during concentration. There is not a lot of literature about this protein and I would appreciate your inputs and suggestions.

Thank you,

Amit