Imidazole in column elution - Removing Imadazole from purified protein without dialysis (Jan/25/2006 )
I was wondering if there are any other methods besides dialysis to remove imidazole from a purified protein. I am currently purifying a protein on Talon's Co2+ resin and eluting with imidazole. The problem is that when I dialyze out the imidazole from my protein, the protein crashes out. The theoretical pI of my protein is around 5.6 and I am using a buffer system that is pH 7.6. Unfortunately, I need this protein for an in-vitro assay so it needs to be active. Imidazole appears to interfere with my in-vitro assay. Also, the reaction that I am doing with my protein requires Mg2+ so I cannot use a chelator of this metal. Any ideas?
-mabbam-
QUOTE (mabbam @ Jan 25 2006, 10:11 PM)
I was wondering if there are any other methods besides dialysis to remove imidazole from a purified protein. I am currently purifying a protein on Talon's Co2+ resin and eluting with imidazole. The problem is that when I dialyze out the imidazole from my protein, the protein crashes out. The theoretical pI of my protein is around 5.6 and I am using a buffer system that is pH 7.6. Unfortunately, I need this protein for an in-vitro assay so it needs to be active. Imidazole appears to interfere with my in-vitro assay. Also, the reaction that I am doing with my protein requires Mg2+ so I cannot use a chelator of this metal. Any ideas?
I had the same problem and I am using Microsep 3K centricons to concentrate and wash thw protein. I have a little lost of protein but at least I get rid of the imidazole
-ana-