What is agarose?

Agarose is a polysaccharide, generally extracted from certain types of red seaweed – Gracilaria and Gelidium.  Agarose is one of the two principal components of agar and is purified by removing its other component, agaropectin.  It is frequently used in molecular biology for the separation of large molecules, especially DNA, by electrophoresis.  However, agarose may also be formed into beads and used in a range of chromatographic methods for protein purification.

For the purposes of chromatography, agarose is widely considered the best material available for protein purification resins.  It is a useful material as it does not absorb biomolecules to any significant extent, has good flow properties, and can tolerate extremes of pH and ionic strength.

 

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

Agarose is an inherently soft material, so we use sophisticated emulsification and crosslinking technologies to ensure excellent pressure flow performance.

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.

Why is agarose used in Ion Exchange Chromatography?

Ion exchange chromatography (IEX) is used to separate molecules based on the strength of their overall ionic interaction with either negatively or positively charged groups on a resin. By manipulating buffer conditions, such as pH and ionic strength, molecules of greater or lesser ionic character can be bound to or dissociated from the solid phase material. IEX supports may either be positively charged (anion binding), or negatively charged (cation binding).

Functional groups such as quarternary amines, DEAE, sulfopropyl or carboxymethyl can be coupled to the agarose, leading to strong or weak anion exchangers, or strong or weak cation exchangers, respectively. For ion exchange chromatography, proteins do not require an affinity tag, making the method suitable for the purification of proteins from natural sources.

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.

Particle size distribution

A narrow particle size distribution offers improved performance characteristics. The more uniform the beads, the more consistently the resin will perform, whether that be when packing the column or running your process.

Most agarose-based resins on the market are produced using Batch Emulsification technology, first utilized around 50 years ago. This produces a relatively wide particle size distribution.

Purolite Life Sciences utilizes a patented manufacturing process called Jetting to produce uniform agarose beads for superior performance. This technology is used in the manufacture of Praesto® Jetted A50 - the only Protein A resin on the market with uniform particle sizes.