Hi!, I am a gratuate student at IMTech., India. I have to use Immobilized metal affinity chromatography. Its adviced that while using imidazole for elution of protein from Nickle affinity column, then one should pre-equilibrate the column with low-molarity of imidazole. Why? Logically, any amount of imidazole will compete with the protein for binding to Ni+. So this seems to be an oxymoron. Please help me understand

A small amount of imidazole in the equilibration buffer discourages nonspecific binding to the column. Assuming your target protein is His- tagged or otherwise has a high affinity for the nickle, the small amount of imidazole in the equilibration buffer will not adversely affect its binding to the column.