Immobilized Metal Affinity Chromatography (IMAC): Competitively elutes 6xHis-tagged proteins from Ni2+/Co2+ resins; 2M Imidazole stock is diluted to ~250–500 mM for elution.

Wash Buffer Preparation for IMAC: Reduces nonspecific binding by adding low imidazole (typically 10–40 mM) prepared from 2M Imidazole.

Binding Buffer Optimization: Uses low imidazole (5–20 mM) to improve selectivity during loading of His-tagged proteins.

Buffering Agent (pH 7.5): Serves as a near-physiological pH buffer for biochemical workflows; 2M Imidazole stock enables precise titration.

Enzyme Assays: Maintains stable pH for pH-sensitive enzyme kinetics in biochemical assays at pH 7.5.

Protein Crystallization: Used as a buffer or additive in screening conditions around neutral pH.

Post-IMAC Processing: Facilitates controlled imidazole gradients for stepwise elution and subsequent buffer exchange/desalting.