protein concentrates on surface of Nickel resin! - (Aug/10/2006 )

Hi to all of my protein friends!

I have encountered a strange problem. My 6xHIS protein has strangely aggregated on top of my nickel HiTrap chelating column. I have tried this step three times with exact results. It is condensed so tightly that I can only get rid of it with 6M GuHCL.

I have tried attempts to drop the protein with usual 500 mM imidazole, low pH, 50mM EDTA, but nothing until GuHCL.

Protein is 40% phobic, 40% charged residues and has a zinc-finger. Mutation is near zinc finger but about 10 aa from site.

Has anyone had similar experiences with a column being surface bound with protein?

Is this a phobic interaction that I'm looking at?

Thanks so much!

Cindy

-biokmst-

hello,

from the sounds of it, your protein is precipitating at high concentrations. i've had similar problems, so i feel your pain. you can try a few tricks to see if they can help, otherwise, it might be worthwhile to switch to a vector system that will give you a fusion protein w/ enhanced solubility characteristics, such as the pMAL-c2 vector family.

depending on your application, you can try a few things:

- add non-ionic detergents like tween 20, np-40, or triton X (or a combination of them)

- glycerol to a max of 5%

- induce your protein at a low temperature, such as 15C for a few hours w/ less than 1mM IPTG

- if you do not need to perform enzyme assays using your purified protein, you can purify it under denaturing conditions...i think Ni columns can tolerate up to 6M urea

i hope this helps.

QUOTE (biokmst @ Aug 10 2006, 10:41 AM)

-johanski-

If you can calculate the pI of your protein based on AA composition, that will help you to chose a pH that gives you the best solubility.

-genehunter-1-