An anion exchange purification (e.g. Q-Sepharose) on a small (e.g. 1x5 mL) column with a 10 CV gradient of NaCl is a pretty good polishing step, and quick. Dilute your protein sample to <20 mM salt and apply to an anion exchange column at pH 8.0 or so, and elute with a 10 CV gradient of 0-0.5 M NaCl or so. That nearly always cleans up partially purified protein preps to crystallography-grade homogeneity. If for some reason you suspect your protein is basic (pI>8.0), try a cation exchanger (e.g. SP-Sepharose) at pH 6.0 or so along the same lines.

Cheers.

On 1/8/2010 7:41 AM, Sivaraman Padavattan wrote:

[log in to unmask]" type="cite">Dear all,
I am trying to express the human protein using bacterial expression strain (Rosetta) and purified using Ni-NTA affinity purification. The Molecular weight of out protein is 47 kDa. In SDS-PAGE, we have seen that 27 kDa contaminant protein co-eluted with our protein even at high concentration of Imidazole. In Superose 12 column, these two proteins eluted as a single peak and its corresponding molecular weight suggestive of partial interaction. By mass mapping we have found 27 kDa band is an adenylate kinase. Is there any specific way to separate adenylate kinase from our protein?
Thanks in advance,

Sivaraman Padavattan

Roger S. Rowlett
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Colgate University
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