Anion-Exchange Chromatography-Based Protein Purification: A Separation Technique to Isolate a Protein of Interest From Dialyzed Bacterial Lysate Based on Net Charge

For anion-exchange chromatography separation of a protein of interest from a dialyzed bacterial protein lysate, add a binding buffer to reduce the lysate viscosity and prevent it from clogging the chromatographic column.

The chelating agents in the buffer bind divalent cations, inhibiting protease activity and preventing protein degradation. Additionally, the buffer with pH above the target protein's isoelectric point, pI, the pH at which the protein has no net charge, causes the excess negatively charged hydroxyl ions to neutralize the protein's positively charged amine groups, rendering protein's surface negatively charged.

Assemble the anion-exchange chromatography column containing cross-linked, agarose-based stationary phase with positively charged quaternary amine groups. Add the binding buffer to prepare the column for maximum protein interaction during sample loading. Load the diluted bacterial lysate.

The negatively charged target proteins bind more tightly to the positively charged groups in the column than less negatively charged proteins.

Run the binding buffer to remove unbound positively charged proteins from the column. Pass an elution buffer through the column, gradually increasing the salt concentration. At low salt concentrations, negative ions compete with weakly bound proteins to bind to the column, causing their elution.

Subsequently, the high salt concentration buffer displaces tightly bound proteins of interest with a longer retention time, eluting the target proteins. The collected target proteins can be used for further analysis.