An Affinity Chromatography Technique for the Purification of a Recombinant Bacterial Protein

Take a suspension of bacterial cells expressing a polyhistidine-tagged target protein.

Sonicate on ice to minimize protein degradation. High-frequency sound waves lyse the cells, releasing intracellular contents, including the target protein.

Centrifuge to separate debris and transfer the supernatant containing proteins into a syringe.

Filter the supernatant to remove aggregates and prevent clogging of the chromatography column.

Take a nickel-based affinity chromatography column and equilibrate it for optimal protein purification.

Load the filtrate onto the column. The polyhistidine tag binds to nickel ions on the column resin, immobilizing the target proteins.

Wash the column with a buffer to remove unbound proteins, and monitor UV absorbance of the eluate to confirm the their removal of non-target proteins.

Add an elution buffer containing imidazole, which competes with the polyhistidine tag for nickel binding, releasing the proteins.

A second spike in UV absorbance confirms the elution of the target protein, which is ready for downstream use.

Sonicate the cells on ice using five cycles of a 15 second pulse and a 30 second pause.

After centrifuging the cell lysate at 21,000 g and four degr