Affinity purification (also called affinity chromatography) is recognized as the most powerful method of purification chromatography or enriching a protein of interest from a complex mixture such as a crude cell lysate, cell culture supernatant, or other samples.
While selective precipitation may be useful in separating different types of macromolecules to some extent, most purification methods rely on the differences in the physical or chemical interactions of the molecules in the solution with a solid, stationary material to achieve the desired results.
In affinity purification, for example, the target molecule is separated from the other component in the solution by treating the solid support with a chemically immobilized ligand that specifically binds with the desired molecule. Thus, as the complex mixture is passed over the column, the target molecules bind to the ligand while the non-bound components in the solution are simply washed away with an appropriate buffer.
Finally, the buffer conditions are altered to break the binding interactions between the ligand and the target molecule and a highly purified target molecule is eluted in the process.
Affinity purification is extremely powerful in that you can purify a sample more than a thousand-fold by passing it once through an affinity column.
Solid Supports for Affinity Purification
Basically, there are two types of solid support used in affinity chromatography: porous gel supports or resins and magnetic beads. However, for this article, we’ll focus mainly on affinity purification resins.
Resin or gel supports are usually polymer resins based on sugar or acrylamide that are produced in solution as 50-150 µm diameter beads. Aside from being extremely porous, the beads are large enough to allow the unimpeded movement of biomolecules around the bead surface as well as through and between the beads. Additionally, the beaded format also makes things easier and more convenient in the lab since they can easily be dispensed to pack columns of any size.
Moreover, since the ligands are covalently attached to the external and internal surfaces of the bead polymer, the sample molecules are allowed to flow freely through a high surface area of the immobilized ligand.
At present, most laboratories use crosslinked beaded agarose for protein affinity purification since it is ideal for gravity-flow, low-pressure procedures, and low-speed centrifugation. Beaded agarose resins can also be subjected to additional crosslinking and chemical hardening to increase their ability to withstand high pressures but be forewarned that this can significantly diminish binding capacity.
This matrix usually comes at 4% (CL-4B) and 6% (CL-6B) densities, both exhibiting medium coupling capacity and are suitable for the same applications (Western blot, high-throughput screening, interaction studies, mutational analysis, functional assay, structural analysis). The size of the beads normally ranges from 45 to 165 µm and they both have a 0.35 MPa pressure limit. They only differ in terms of exclusion limit (20,000 kDa for CL-4B and 4,000 kDa for CL-6B).
In addition to agarose gels, polyacrylamide-based resins can also be used for column affinity chromatography. While both mediums exhibit low non-specific binding characteristics, the polyacrylamide-based resin is not easily compressed and can withstand medium-pressure applications.
G-Biosciences offers a wide range of affinity purification kits and resins to suit a wide variety of applications. Here are some examples that can help you with your affinity purification challenges.
- Immobilized biotin and iminobiotin resins for avidin-streptavidin and neutravidin purification
- Calmodulin resin for purifying calmodulin-binding proteins (CBP)
- Immobilized p-Aminophenyl Phosphoryl Choline and the more cost-effective immobilized O-Phosphorylethanolamine for the purification of C-reactive proteins
- GST-tagged or His-tagged protein purification kits or resin
- Immobilized heparin and lectins for isolating lipoproteins, growth hormones, hemostatic proteins, and other physiological markers
- Thiopropyl resin for purification of thiol group containing proteins.
Download the Purification Resins Handbook and Selection Guide for a full list of products.