Question

Explain in details the IMAC, it's concept column matrix, washing and Elution buffer

Answer 1

IMAC - Immobilized metal affinity chromatography

IMAC is a separation technique, which is used to separate various proteins based on metal ions, with which they form coordinate bonds. Mainly, recombinant proteins having poly-His ends are separated by this technique. The function of these histidine tags is to form coordinate bonds with metals. This coordinate bond between protein and metal is irreversible in nature.

Column matrix - The matrix in columns of IMAC is made up of either cobalt, zinc, copper, or the metal that can form a coordinate bond with the protein's His-tags. These metals get attached to the matrix and bind to the protein via the imidazole rings found in histidine.

Washing buffer - This buffer helps in washing away the unwanted proteins that weakly bind to the matrix. This buffer is made up of low concentrations of imidazole with 6.3 pH.

Elution buffer - In this step, the protein is desorbed from the matrix. This is done by putting a more competitive binder than Histidine in the column matrix. This molecule will replace the imidazole ring of histidine and will bind to the metal in the matrix. The buffer used here has pH between 4.5 and 4.7.