If I were you, I would run another ion exchange column at a pH about 2 units lower than the protein's pI. If necessary, you have other options, such as size-exclusion chromatography or hydrophobic interaction chromatography.

You can add a step before your purification with this protocol :

- Protein Precipitation Using Ammonium Sulfate

10.1002/0471140864.psa03fs13

Screening the quantities of selected ammonium sulfate will allow you to precipitate the protein contaminants that you do not want and, or your protein in order to improve its purity. Use the tables to help you.

check out this publication from JBC: Purification and Preliminary Characterization of a Serine Hydrolase Involved in the Microbial Degradation of Polychlorinated Biphenyls

Hi Ana Cruz ,

doing two (similar) ionex chromatographic steps is a little pointless. You need to find another dimension, where to resolve your proteins. For example, size exclusion chromatography, hydrophobic interaction chromatography (C4 or C8 columns) or some affinity chromatography. It may be helpful even if your protein will not bind, if other proteins will bind (but of course it's better if your protein binds, because then you can play with the elution).

Alternatively, you can do some crude separation method in the beginning like ammonium sulphate, manganese or protamine sulphate precipitation or so isoelectric focusing at the end to get specifically your protein (but its usability depends on what you want to do with your protein, i.e. how much you need of it).

BTW when I was purifying my protein to identify it, I used like 5 or 7 columns.

Please check your SDS-PAGE, method of sample preparation, possibility of multimerization and artifactual bands.