 Solution

The compound imidazole resembles the metal-coordinating part of the histidine side chain and competes for binding to the nickel sites in immobilized metal affinity chromatography. At the lower concentration in the wash, it helps displace weakly or non-specifically bound proteins while the multiple contacts of the histidine tag retain the desired enzyme. At the higher elution concentration, competition is strong enough to displace the tagged enzyme. Low imidazole improves washing; high imidazole elutes the target. Remove or match the elution-buffer components before interpreting downstream assays, and verify purity independently of total protein concentration.