Other proteins are stable only above or below their pI. These proteins can be purified with either an anion or cation exchanger. Some proteins are stable both above and below their pI. In these scenarios, the selection of an anion or a cation exchanger depends on the protein of interest’s stability. The latter three matrices allow higher flow rates.ĭeciding between an Anion Exchanger and a Cation Exchangerįor many protein purification workflows, protein folding and stability is a concern. Common matrices are cellulose, agarose, polymethacrylate, polystyrene, and polyacrylamide. Ion exchange chromatography resins are composed of positively or negatively charged functional groups that are covalently bound to a solid matrix. For these proteins, elution with a pH gradient may not be possible. Note: Some proteins fall out of solution at a pH equal to their pI. Three overlaid chromatograms showing how changing pH from 6.5 to 8.5 shifts the elution profile when eluting using a salt gradient.
Altering the pH of the elution buffer can affect the resolution of the method:įig. Lastly, pH can be used to refine elution when using a salt gradient.
Since it is very difficult to generate reproducible and accurate linear pH gradients, a step-gradient is generally chosen when pH is used for elution. Elution of protein ( blue trace) with an increasing pH gradient ( red trace). To elute proteins from an anion exchange resin, a decreasing pH gradient is chosen, while an increasing pH gradient is chosen for elution from cation exchangers.įig. Proteins will elute when the pH gradient reaches their pI, because they will no longer carry a net charge that allows them to interact with the column resin. Here, a pH gradient is chosen that approaches the protein of interest’s pI. Proteins with few charged groups will elute at low salt concentrations, whereas proteins with many charged groups will have greater retention times and elute at high salt concentrations.Īlthough less common, a pH gradient can also be used for elution. The charged salt ions compete with bound proteins for the charged resin functional groups. Elution of proteins ( blue trace) with an increasing salt gradient ( red trace). Cation exchanger - 0.5–1.5 pH units less than the pI of the protein of interestĪfter loading an impure protein sample onto an ion exchange chromatography column, the column is washed to remove undesired proteins and other impurities, and then the protein(s) of interest is eluted using either a salt gradient or a change in pH.įig.Anion exchanger - 0.5–1.5 pH units greater than the pI of the protein of interest.A good rule of thumb for choosing a buffer pH is the following: For example, if an anion exchange resin is chosen, all proteins that are negatively charged at the loading buffer pH will bind to the positively charged column resin. When an ion exchange chromatography column is loaded with a sample at a particular pH, all proteins that are appropriately charged will bind to the resin. In a buffer with a pH lower than the pI of the protein of interest, the protein will carry a positive net charge thus a negatively-charged cation exchange resin is chosen. In a buffer with a pH greater than the pI of the protein of interest, the protein will carry a net negative charge therefore, a positively charged anion exchange resin is chosen to capture this protein. The choice of buffer pH then determines the net charge of the protein of interest.
The pI value can be calculated based on the primary sequence of the molecule.
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