Column chromatography is an established technique that has been adapted to purify recombinant proteins, monoclonal antibodies, and other biomolecules from complex mixtures. To achieve this, a relevant binding partner is immobilized on to agarose beads, which are then packed into a column to function as an affinity resin. Once the target of interest has been captured on the beads, it can be eluted for further downstream analysis. For this method to be successful, it is vital that the chromatography column is packed and run appropriately. This article discusses effective ways of accomplishing this and highlights the advantages of choosing pressure over gravity for separation.
Although a wide variety of pre-packed columns is commercially available, it is often more cost-effective to pack your own columns by purchasing a specialized affinity resin supplied in a loose resin format. Be sure to select a suitable resin to bind your target (e.g. glutathione agarose resin to purify GST-tagged proteins from cell growth media, Protein A agarose resin to purify antibodies from sera) and check the product datasheet to understand the resin’s binding capacity and therefore the volume of beads needed, as well as the pressure the resin can withstand.
When choosing an empty column for packing in-house, consider the type of purification technique you will use. While low-pressure columns are usually appropriate for manual purification methods, purifications that rely on Fast Protein Liquid Chromatography (FPLC) design systems will require columns able to tolerate higher pressures. Narrow columns typically allow a higher pressure than wide columns, however the maximum flow rate achievable will depend not only on the column diameter but also on the buffer viscosity.
Air bubbles can be catastrophic to column chromatography, blocking sample flow and necessitating that the entire procedure is started over. Not only does this waste time but it can also result in a significant loss of material. To avoid introducing air bubbles when packing the column, bring the resin and any buffers to room temperature and de-gas them for at least 30 minutes before use. Shaking the beads gently will ensure they are a homogeneous suspension before they are added to the column, while pouring them slowly down the column walls is essential to accomplish a uniform distribution. Never use a pipette for bead addition as pipettes can easily become blocked.
It is important to check the column performance before starting the purification process, and also between runs to confirm there have been no changes to the integrity of the bead bed. There are several ways to do this, the most common being to pass a tracer substance through the column to determine the theoretical plate number and peak symmetry. Critical parameters that can affect the column efficiency include the buffer viscosity, agarose bead diameter, column diameter, column pressure, and flow rate.
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Various factors have the potential to influence target binding to the agarose beads. It can therefore be helpful to perform a literature search before carrying out a column chromatography procedure to optimize parameters such as pH, temperature, or salt/ion concentrations to suit your specific biomolecule. For instance, working at lower temperatures and adding protease inhibitors to buffers can help to minimize protein degradation, while diluting a sample 1:1 with binding buffer before adding it to the column is an effective way to maintain the proper ionic strength and pH for optimal binding.
While gravity-flow column chromatography can easily be performed without the need for specialist instrumentation, it is limited by more labor-intensive workflows, considerably longer timelines, and inferior reproducibility compared to FPLC. Gravity-flow systems also introduce an increased risk of beads drying out, for example if a stopper cap should fail or the end-user is called away from the column unexpectedly.
In contrast, by employing pre-programmed protocols and a walk-away methodology, FPLC provides extremely tight control of flow rates during column packing and chromatography separation, translating to highly reproducible performance. Additional advantages of FPLC are that its wide flow range makes it compatible with many different column types, and it also allows for multiple columns to be run in parallel. Moreover, FPLC can be easily coupled to other in-line instrumentation to further streamline workflows.
Agarose Bead Technologies (ABT) offers a wide range of high-quality agarose beads for column chromatography applications. More information can be found here.