Gunnar Glad

Separation of DNA restriction fragments by ion-pair chromatography.

The separation of restriction endonuclease fragments of DNA on columns of Pharmacia PepRPC (C2/C18) has been studied. The effect of different concentrations of triethylammonium or tetrabutylammonium salts as ion-pairing reagents, as well as of physical parameters, such as flow-rate and sample load, has been investigated. With the use of triethylammonium buffers, removed by evaporation under vacuum, separated fragments were recovered in yields of 68%. Isolated fragments were accessible to further cleavage with restriction enzymes. Resolution of fragments ranging from 10 to 3000 base pairs depended primarily upon molecular size.

A novel and conserved pocket of human κ‐Fab fragments: Design, synthesis, and verification of directed affinity ligands

Antibodies of type IgG may be divided into two classes, called λor κ, depending on the type of light chain. We have identified a conserved pocket between the two domains CH1 and CL of human IgG κ‐Fab, which is not present in the λ type. This pocket was used as a target docking site with the purpose of exploring the possibilities of designing affinity ligands that could function as such even after immobilization to gel. The idea of the design arose mainly from the results of the saturated transfer difference (STD‐NMR) screening of 46 compounds identified by means of virtual docking of 60 K diverse compounds from the Available Chemicals Directory (ACD). Surface plasmon resonance (SPR) was used as an alternative method to monitor binding in solution. A total of 24 compounds belonging to a directed library were designed, synthesized, and screened in solution. They consist essentially of an amino acid condensed to a N,N′‐methylated phenyl urea. STD‐NMR results suggest that a small hydrophobic side chain in the condensed amino acid promotes binding, whereas a hydroxyl‐group–containing side chain implies absence of STD‐NMR signals. Three compounds of the directed library were immobilized and evaluated as chromatographic probes. In one case, using D‐Pro as the condensed amino acid, columns packed with ligand‐coupled Sepharose (Amersham Biosciences) retained two different monoclonal samples of κ‐Fab fragments with different variable regions, whereas a sample of monoclonal λ‐Fab fragments was not retained under similar chromatographic conditions.