The use of Dyes in Affinity Ligand and Protein Immobilization

Abstract

We have been using heterocyclic dyes to activate hydroxylic matrices, such as paper or Sepharose (agarose), for subsequent covalent attachment of ligands or proteins through a secondary amine (lysine) or thioether (cysteine) linkage. The activation provides a colored matrix whose coupling to ligands can be followed by the loss of color. The dye can also serve as an affinity ligand for the affinity-directed immobilization of a protein. In the latter case, the protein is attached to the matrix in a specific way, due to its affinity for the dye. The affinity-directed immobilization of IgG through its Fab segment is being used as a model system.

As an example of the chromophoric aspect, the activation of paper with FMNI (3-fluoro-2-methyl-5-[2-naphthol-l-azo]-indazolium tosylate) and the coupling of several proteins is discussed.

As an example of affinity-directed immobilization, the coupling of anti-DNP antibodies to DNP-CMP (5-amino-2-chloro-N-[2,4-dinitrophenyl]-l-methylpyridinium tosylate) activated Sepharose is discussed.