Troubleshooting

Problem	Potential Cause and Solution
Protein does not bind the cation/anion exchange column	(1) The wrong ion exchange column is being used. Assess the pI (and pH of the loading buffer) and confirm that the domain of interest will bind to the chosen column. (2) The salt concentration in the loading buffer is too high. Exchange into a lower salt buffer before loading
The protein has the incorrect retention volume on gel filtration based on expected for the molecular weight	Run standards in the chosen buffer to confirm column performance and where the protein should be eluting. (1) The protein may have degraded. Check by SDS-PAGE and alter conditions to reduce degradation. (2) The protein may be unfolded. This can be confirmed by another method such as circular dichroism. Construct boundaries may need to be altered to obtain a well-folded domain, or expression/ purification conditions may need to be altered to promote folding. (3) The protein is aggregating. Alter buffer composition to decrease aggregation