A conjugation chemistry that introduces a charged functional group into the support can cause nonspecific binding by promoting ion-exchange effects. A linkage that allows the coupled ligand to leach from the matrix will result in contamination of the purified protein and shorten the useful life of the affinity support. For example, if the linkage blocks or adversely affects the structure of the immobilized ligand, it will limit its effectiveness for affinity purification. The type of linkage that is formed between the matrix and the immobilized ligand affects the performance of the affinity support in a number of ways. The activated complex then can generate a covalent linkage between the ligand and the support, resulting in ligand immobilization. Usually, the solid phase matrix first is activated with a compound that is reactive toward one or more of these functional groups. The types of functionalities generally used for attachment include easily reactive components such as primary amines, sulfhydryls, aldehydes, and carboxylic acids. Regardless of the intended application, the chemical reactions that make possible ligand attachment are well characterized and facilitate the attachment of biomolecules through their common chemical groups. Applications of affinity targeting include purification, scavenging (or removal of contaminants), catalysis (or modification of target molecules) and a broad range of analytical uses to quantify a target molecule in a sample solution.ĭesigning custom affinity supports that are able to target unique biomolecules requires methods to covalently link a ligand to an insoluble matrix. Affinity ligands are now coupled to magnetic particles, latex beads, nanoparticles, macro-beads, membranes, microplates, array surfaces, dipsticks and a host of other devices that facilitate the capture of specific biomolecules. The concept of using immobilized affinity ligands to target biomolecules has extended beyond just chromatographic applications with beaded agarose resins (still the most common). The target protein is eluted in a purified and concentrated form. Elution conditions may be specific, such as a competitive ligand, or nonspecific, such as changing pH, ionic strength, or polarity. The bound protein is then recovered in a highly purified form by changing conditions to favor elution. The protein of interest is tightly bound to the resin under conditions that favor specific binding to the ligand, and unbound contaminants are washed off. Affinity chromatography is very selective and provides high resolution with an intermediate to high sample loading capacity. Small scale affinity purification using an antibody immobilized to a solid support. Chromatography has three main components: the mobile phase or solvent containing proteins, the stationary or solid phase also called the medium or resin (which may be agarose or other porous resin) and the chromatography column. The affinity ligand can be any molecule that will bind the target without also binding other molecules in the solution. A ligand having affinity for a target molecule is covalently attached to an insoluble support and functions as bait for capturing the target from complex solutions. Examples include Protein A agarose resin for general antibody purification and streptavidin magnetic beads for purifications involving biotinylated molecules.Īffinity chromatography utilizes the specific interactions between two molecules for the purification of a target molecule. Affinity ligands that have broad applicability are commercially available in a variety of ready-to-use, pre-immobilized forms. Many assay methods, such as ELISA and western blotting, depend upon strong but noncovalent immobilization of antibodies or other ligands to the solid material (polystyrene microplates and nitrocellulose membrane, respectively).īy contrast, most affinity purification strategies – especially those involving antibodies and other proteins – depend upon covalent chemical conjugation of ligands to the solid support matrix. The latter class of molecules are called affinity ligands, and they can be attached (immobilized) to the solid material (stationary phase) in a variety of ways. Affinity purification and many assay techniques depend on the binding interactions of target biomolecules in liquid samples with specific chemical groups or macromolecules that are immobilized on a solid material.
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