Eluting the protein from a Proteus IMAC spin column

The most common elution conditions for IMAC separations involve the use of a competitive counter-ligand such as imidazole. This is the preferred elution method for purifications under native conditions. For purifications under denaturing conditions, elution is performed either using imidazole in the presence of denaturant such as 8 M urea or by a reduction in elution pH from pH 7.4 to pH 4.5. It is important to appreciate that a few proteins are acid-labile and they can lose their activity at very low pH values. Above all, the elution conditions must preserve the integrity and activity of the target protein. Most observed denaturation is caused by harsh elution conditions.