Protocol Online logo
Top : New Forum Archives (2009-): : Protein Expression and Purification

Compatibility of Urea and Imidazole - (Aug/25/2013 )

I would like to make a buffer containing 8 M Urea, 250 mM Imidazole and 1 M NaCl; but I am not sure if Urea and Imidazole can be in the same buffer. Hence, I would greatly appreciate your feedback on this.


Background info:

I am purifying a protein using affinity chromatography (N-term Histag, cleavage with thrombin). And I would like to merge the last two steps of my current protocol, namely removal of cleaved Histag fragment and of thrombin. My idea is to pass my overnight cleaved sample through both nickel and benzamidine columns in series, hence leaving me with pure protein in the flow-through. After that I would like to remove the Histag fragment from the Nickel column and the thrombin from the bezamidine column. Thus, I thought about using a Tris buffer (pH 7.5 adjusted with HCl) with 8 M Urea, 250 mM Imidazole and 1 M NaCl; since it should remove everything that is bound by affinity and ionically. However I do not know the compatibility of Urea and Imidazole, indeed I never used Urea before.


there should be no problem mixing urea and imidazole.


keep in mind that imidazole is a buffer and addition of tris may not give you the pH you want (you may have to adjust).


Thank you a lot for your comment. smile.png