5 replies to this topic

[Malfet]

Hi all!

I'm trying to couple my proteins of interest to BrCN-sepharose. I have two proteins with very similar sequences. They are expressed in E. coli, purified on NiNTA-column through his-tag, eluted with 450 mM Imidazol and dialyzed against coupling buffer. All these steps are always fine, without any troubles, however when I add my proteins to the BrCN-activated sepharose they precipitates. My ordinary coupling buffer is standard 0,1M NaHCO3 with 0,5M NaCl (pH=8,3). I've tried to perform coupling in PBS (pH=7,4) plus 0,5M NaCl as a buffer with pH further from the protein's isoelectric point (around 9), but with the same outcome. Perhaps, even worse. 5% glycerol does not help either. Coupling at room temperature for 3-4 hours instead of doing it overnight in the cold room helps, but just a little bit. One protein is almost fine at room temperature, however second protein precipitates anyway. I've also changed the water and used different Br-CN sepharose batch.
Any help would be precious.

Edited by Malfet, 22 January 2010 - 07:28 AM.

[mdfenko]

you could try reducing the nacl concentration, maybe to ~0.2M. high salt may be causing the precipitation.

[Malfet]

mdfenko, on Jan 23 2010, 05:17 AM, said:

you could try reducing the nacl concentration, maybe to ~0.2M. high salt may be causing the precipitation.

Thank you for the answer. I don't think so, because I have the same NaCl concentration in all buffers (lysis, wash, dyalisis buffers) and proteins are stable there... well, only if in the beads presence proteins are more sensitive to high salt, but I don't have such problem with Ni-beads step...

[mdfenko]

you used 5% glycerol? shouldn't that have been ethylene glycol?

Edited by mdfenko, 25 January 2010 - 07:18 AM.

[Malfet]

mdfenko, on Jan 25 2010, 03:18 PM, said:

you used 5% glycerol? shouldn't that have been ethylene glycol?

Why? Glycerol I used just as a reagent which sometimes helps to improve protein stability, but it did not help. Course, ethylene glycol can also do that, but do you think it will act significantly better?

[mdfenko]

glycerol is used to prevent freezing and to prevent ice crystal formation thet may damage your protein. ethylene glycol (antifreeze, but not in this case) is used to enhance the hydrophilicity of a protein (it is used to enhance elution from hydrophobic interaction chromatography).