After purification of His-tagged recombinant protein using a nickel column, the bands are impure. How can I optimize the conditions?

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Don't rush to use molecular sieve chromatography yet; at the nickel column stage, widening the wash condition differences makes it easier to suppress contaminant bands. It is recommended to add 20 mM imidazole and 300 mM NaCl to both the lysis buffer and the equilibration buffer. After loading, first wash with 30 mM imidazole for 8-10 column volumes, then with 50 mM imidazole for 3-5 column volumes, and finally elute with 250 mM imidazole. If the contaminant bands are washed off at low concentrations, this indicates nonspecific binding, and you can reduce the loading amount or extend the equilibration time; if the contaminant bands mainly co-elute with the target protein at 250 mM, then consider gradient elution at 100/200/300 mM to see whether the contaminant bands can be separated from the 28 kDa target band.

Whether to add size exclusion chromatography depends on the source of the contaminant bands. First, run SDS-PAGE to observe the size of the contaminant bands: those significantly smaller than 28 kDa are mostly degradation fragments, and it is recommended to add PMSF/protease inhibitors and shorten the purification time; those larger than 28 kDa are often host proteins or nucleic acid complexes, and an anion exchange step can be added, which is faster and lower in cost than size exclusion. If the contaminant bands are close in molecular weight, then consider size exclusion such as Superdex 75. With a budget of under 500 yuan per run, prioritize fine elution on a 1 mL Q/SP column, which can usually remove most host proteins; size exclusion resins and prepacked columns easily exceed the budget.

Many nonspecific bands actually come from incomplete lysis and nucleic acid contamination. For 200 mL of bacterial culture, it is recommended to disrupt by sonication or high-pressure homogenization, then add a small amount of Benzonase nuclease together with 1 mM PMSF to reduce the viscosity of the lysate before centrifugation. Centrifuge at 16,000 g for 20 min, collect the supernatant, filter through a 0.45 μm membrane, and load onto the column. If the target protein exists as inclusion bodies, wash the inclusion bodies first and then perform denaturing nickel column purification; nonspecific bands will be greatly reduced. After elution, measure A280 and confirm by electrophoresis before deciding whether the next step is needed. These upstream optimizations do not cost much, but they are often more effective than increasing imidazole.