Michael J. Harvey

The potential of Ultrogel®, an agarose-polyacrylamide copolymer, as a matrix for affinity chromatography

Received 13 March 1976 1. Introduction The selection of the inert support in the synthesis of adsorbents for affinity chromatography is constrain, ed by several factors. It should be stable to chemical and biological degradations, hydrophilic, easily substituted and uniform to enhance the chromato- graphic process. The comparative advantages of polyacrylamide, glass, cellulose, cross-linked dextrans and beaded agarose have been reviewed [1-3] ; currently the most popular support materials are polysaccharides, especially beaded agarose. Recently the microscopic structure of these polysaccharide matrices has been investigated both with respect to their ability to withstand various activation procedures, in particular cyanogen bromide [4], and also the distribution of the immobilised ligand throughout the matrix following coupling [5,6], which in itself may be a function of the CNBr activation [7]. In a previous communication [4] we reported that beaded cross-linked dextrans were susceptible to structural damage during CNBr activation, whilst beaded agarose was not obviously affected provided that vigorous stirring was avoided. However, microscopic inclusions and vacuoles were observed in approx. 5% of the commercially available agarose beads. Subsequently Gribnau et al. [8] reported the presence of similar particles in commer- cial preparations of agarose. The uncertain effect of

[17] N6-immobilized 5′-AMP and NAD+: Preparations and applications

Publisher Summary The use of group-specific adsorbents, in particular immobilized cofactors, permits the examination of a whole range of enzymes in a single affinity system. The chemical complexity of polyfunctional cofactors has, until recently, restricted the potential methods available for their immobilization. Nevertheless several preparations of chemically defined polymers are now documented.

An Analysis of Affinity Chromatography Using Immobilised Alkyl Nucleotides

Group specific matrices for affinity chromatography have now received considerable attention (1–9). Some of these studies have provided amongst the best examples of this aspect of affinity chromatography (10). Amongst the group specific matrices and in particular amongst the immobilised cofactors, the nicotinamide nucleotides present a very wide choice of applications and of different enzyme systems to study. However, because of the chemical complexity of the nicotinamide nucleotides, severe restrictions are imposed on the potential methods available for their immobilisation. These nucleotides may be immobilised readily onto cellulose or Sephadex or agarose containing aminocaproic acid (1,11,12). The resultant polymers are almost certainly of undefined nature (13) apart from being contaminated with unreacted carboxyl groups. The former need not necessarily be a drawback; the polymer preparation described by Allen & Majeries (10) does not specify which carboxyl groups of B1 2 are utilised in the linkage to the agarose and this undefined matrix is capable of purification factors of thousands. This is not so with nucleotides where the purification factors rarely exceed fifty times (1,8).