This article details the process of anion-exchange chromatography for separating proteins from bacterial lysates. It emphasizes the importance of buffer composition and pH in optimizing protein binding and elution.
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.
Start the FPLC with general maintenance. Connect column buffers AIEX-A and AIEX-B to buffer valves, and the anion-exchange resin-containing column to the column ports of the FPLC. Adjust the general chromatographic parameters accordingly.
Start the AIEX program in the FPLC software and choose the equilibration volume for buffer A. Subsequently, load the sample onto the column, and elute the proteins with a linear gradient from 0% to 100% high-salt buffer. Collect 2-milliliter fractions during elution.
Analyze the eluted fractions after finishing the run.
繁體中文
