Activated media for ligand immobilization allow the researcher to create an affinity matrix of choice by coupling a ligand such as an antibody, enzyme, antigen, or receptor. There are multiple coupling methods. Three methods will be discussed here.
Related Topics: Affinity Chromatography, Affinity Purification of Tagged Recombinant Proteins, and Protein A/G Affinity.
Cyanogen bromide activation is the most common method for preparing affinity gels. It reacts with the hydroxyl groups on agarose supports to form cyanate esters and imidocarbonates. These groups react with primary amines to couple the protein onto the agarose matrix. Because cyanate esters are more reactive than are cyclic imidocarbonates, the amine will react mostly with the ester, yielding isourea derivatives, and partially with the less reactive imidocarbonate, yielding substituted imidocarbonates.
Disadvantages include the toxicity of cyanogen bromide and its sensitivity to oxidation. Cyanogen bromide activation also involves the attachment via an isourea bond, which is positively charged at neutral pH and thus unstable. Consequently, isourea derivatives may act as weak anion exchangers.
Ligands containing amino, thiol, or hydroxyl groups can be coupled to the epoxide through an epoxy ring-opening reaction under mild conditions. Following immobilization, active groups remaining on the resin need to be deactivated or blocked to avoid undesirable reaction with proteins of interest during affinity chromatography. They can be deactivated or blocked by mixing the resin with 1 M ethanolamine, pH 8–l9.
This reaction mechanism involves two steps: the activation of carboxyl groups with displacement by the nucleophile, R-NH2, thereby releasing the EDAC as a soluble urea derivative. The carboxyl groups can be present on the ligand or on the matrix. The activated carboxyl groups react with the amino groups in the reaction mixture. The illustration shows the EDAC coupling reaction for a ligand with a terminal carboxyl group to Bio-Rad’s Affi-Gel® 102 gel.
Bio-Rad's activated affinity chromatography media.
N-hydroxy- succinimide µmol/ml ≥10 µmol/ml
There are a number of preactivated media that include dyes, metals, and other ligands. The correct choice is dictated by the group available in the ligand molecule and the nature of the binding interaction with the molecule to be purified. High selectivity and capacity make this technique ideally suited to the isolation of specific components from complex biological mixtures.
Bio-Rad's ready-to-use activated affinity chromatography media.
* Refer to bulletin 3193 for purification conditions. ** For example, Profinity GST resin is available only in prepacked cartridges. ***Profinity eXact purification resin.
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