Glyoxyl agarose is constituted by quite thick agarose fibres containing a large number of very stable aldehyde groups attached to the support by very short spacer arms. Under alkaline conditions, these activated supports immobilize proteins, via, at least, a two-point reaction involving the region/s of the protein surface with the higher densities of amino groups. These bonds are weak Schiff's bases and the reversibility of the bonds has been used to convert this matrix into a chromatographic one. A more intense multipoint attachment between the immobilized protein and the activated support can be further promoted, with minimal loss of catalytic activity, by a long-term incubation of the protein-support conjugate under suitable conditions. The end-point of the preparation of agarose-protein conjugates is a very mild borohydride reduction. After that reduction, the enzyme remains attached to the support by means of very stable secondary amino bonds (with very similar physical properties to those of the former primary amino ones) and the remaining aldehyde groups on the support are converted into fully inert hydroxyl groups. Very active and highly stabilized derivatives of many enzymes and proteins have been prepared using these supports. The main features of these protein immobilization protocols are discussed here. (c) 2005 Elsevier Inc. All rights reserved.