Protein aggregation after purification: Imidazole?

Lisa Kueltzo kueltzo at ku.edu
Fri Sep 28 11:23:12 EST 2001


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.  I am planning a step dialysis, slowly remiving
the imidazole while maintaining the ionic strength with NaCl, and then
slowly dropping the NaCL and phosphate down to the desired
concentrations.

Does anyone have any insights or suggestions about the aggregation or
my proposed solution?  Thank you in advance for any help you can give
me.  Fell free to reply by post here or email.

Thanks,
Lisa Kueltzo




More information about the Methods mailing list