Before you can study your protein’s structure or function, you need to get it pure enough for your application. The liquid chromatography methods frequently used to purify proteins have the same basic procedure – flow the protein-containing solution through a column packed with a chromatography resin. But how you do that and the tools you use can affect your results and efficiency.
How liquid flows through a column
The way that liquid flows through a chromatography column depends on how it is packed. A well-packed bed generates a stable column that offers good resolution. An even flow of liquid through the column will generate peaks that are narrow and sharp, as shown in Fig 1B.
A poorly packed bed results from a non-homogenous slurry. This generates a less stable bed and an uneven flow through the column. Peaks will be broad, as shown in Fig 2B.
A well-packed column ensures reproducible column performance run after run. To get good purification results, especially for size exclusion chromatography (SEC), a well-packed column is critical. When liquid moves unevenly through the column, protein peaks will be broad and might overlap with other neighboring peaks. Therefore, the purity of the target protein could be negatively impacted when using a poorly packed column.
Many vendors offer a variety of prepacked columns for small-scale purification. The benefit with prepacked columns is that the packing method has been carefully optimized, and the packing is always carried out in the same way. With manual packing, the result tends to vary.
All prepacked columns from GE Healthcare are tested before release. Also, every prepacked HiLoad and Tricorn SEC column is tested individually with regards to number of theoretical plates (N/m).
Packing a column can be both difficult and time-consuming and needs expertise. For SEC, columns can take up to a full day to pack. For other techniques, packing can require over an hour (see figure).
Instead of spending hours of your time packing and testing your column before you can start to purify your protein, consider purchasing a manufactured prepacked column. This will save you time, and you can be sure that you will have a well-packed column that you can start using immediately.
If the prepacked column you need is not available as a regular product, contact our Custom Products group. We can customize columns and resins to your specific requirements.