What is agarose?

Further advantages of using agarose for chromatography include:

• Extremely hydrophilic – minimal unspecific binding
• Low matrix volume (4-8%) – possible to achieve high capacity
• Easy to conjugate
• Very stable under alkaline conditions

Why is agarose used in Affinity Chromatography?

Affinity chromatography is a method for selective purification of a molecule or group of molecules from complex mixtures based on highly specific biological interaction between the two molecules. The interaction is typically reversible and purification is achieved through two phases, with one of the molecules (the ligand) immobilized to a surface while its partner (the target) is in a mobile phase as part of a complex mixture. The capture step is generally followed by washing and elution, resulting in recovery of highly purified protein. Highly selective interactions allow for a fast, often single step, process, with potential for purification in the order of several hundred to thousand-fold.

For affinity chromatography, beaded agarose is the most commonly used matrix resin for the attachment of the ligands that bind protein.  The ligands are linked covalently through a spacer to activated hydroxyl groups of an agarose bead polymer. Proteins of interest can then be selectively bound to the ligands to separate them from other proteins, after which it can be eluted. The agarose beads used are typically of 4% and 6% densities with a high binding capacity for protein.

Does size matter?

Agarose resins come with different particle sizes and size distributions. These have an impact on the physical properties of the purification matrix.

• Pressure - the smaller the beads, and the narrower the size distribution, the higher the pressure-resistance of the beads. In comparison, the larger the beads, the faster the flow rate in batch and FPLC experiments.

• Binding capacity - the smaller the beads, the higher the ratio of surface to volume, and the higher the binding capacity of the beads. Although binding capacity is also dependent on other factors such as the type and size of ligand, and density of ligand coupling.