FAQ: How can I reduce contaminating proteins in a protein purification when using Ni Resin?

Add up to 10 mM imidazole to the Lysis buffer to prevent contaminants from binding (optimal imidazole concentrations must be determined empirically). Employ extra wash steps (e.g., up to 5 additional column volumes) with the recommended wash buffer. Increase the imidazole concentration in the wash buffer (test by increasing in 5 mM increments up to 20 mM); higher concentrations of imidazole can result in decreased overall yield of the target protein. The optimal concentration of imidazole will be target/impurity dependent and will therefore require optimization.  

If you suspect contaminants are associated with the His-tag fusion protein, add 10 mM β-Mercaptoethanol or 1 mM THP to reduce disulfide bonds. Increase salt and/or detergent concentrations or add ethanol/glycerol to the wash buffer to disrupt non-specific interactions.

Contaminants can also arise as truncated forms of the His-tag fusion protein, check for possible internal translation starts (C-terminal His-tag) or premature termination sites (N-terminal His-tag). Avoid protein degradation during the purification by keeping the resin cold (4°C) or by including protease inhibitors.