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.