FAQ: How can I reduce contaminating proteins in my Ni-NTA His-tag protein purification?

It is important to ensure that the beads are fully re-suspended during wash steps. Use end-over end inversion or use a benchtop shaker set at 850 rpm to ensure thorough mixing during the wash steps. employing extra wash steps (e.g., up to 5 washes) with the recommended wash buffer can sometimes improve purity. Increasing the imidazole concentration in the wash buffer (test by increasing in 10 mM increments) may increase purity but can sometimes result in decreased overall yield of the target protein. The exact concentration of imidazole will be target/impurity dependent and will therefore require optimization. Addition of detergents such as Triton X-100 and Tween 20 (0.05-0.1%) in the lysis, wash and elution buffers can often reduce nonspecific binding. Employing a tag with 7-8 histidines may allow for higher imidazole (up to 50 mM) washes and better target purity. Increasing the NaCl concentration (up to 1M) in the wash buffer may reduce the binding of contaminants as a result of nonspecific ionic interactions.