Protein aggregation after purification: Imidazole?

D.K. dk at
Fri Sep 28 18:59:48 EST 2001

kueltzo at (Lisa Kueltzo) wrote:
>After purifying a soluble, recombinant his tag protien on a Hi-trap
>Nickel chelating column, I dialyze the protein from the elution buffer
>(165mM Imidazole, 300mM NaCl, 50mM Phosphate, pH8.0, ionic strength
>0.54) to my experimental buffer (10mM Phosphate, pH7.4,200mM NaCl,
>ionic strength .23), the protein aggregates extensively.  There is
>still some soluble protein, and the agggation appears reversible by
>high amounts of NaCl.  I was wonding if the removal of the imidazole
>is the key problem.  

Imidazole, or its removal, cannot have anything to do with it. 

In descending order of likelyhood: 

- your protein (at least its 6His form) requires high salt for solubility;
- tends to aggregate at pH < 8;
- aggregates in the presense of phosphate;
- any combination of the above. 

>Does anyone have any insights or suggestions about the aggregation or
>my proposed solution?  

If you must use conditions with lower salt, lower pH and phosphate, then
you can try adding detergent - it may prevent aggregation.


More information about the Methods mailing list