The Immobilized Ligand

Porous glass (PG) modified with covalently adsorbed poly(p-nitrophenyl acrylate), as described in Sect. 4.1, turned out to be a highly suitable carrier for immobilization of various biospecific ligands and enzymes. When the residual active ester groups of the carrier were blocked by ethanolamine, the immobilized ligands when bound to the solid support via hydrophilic and flexible poly(2-hydroxyethyl acrylamide). The effective biospecific binding provided by the ligands... 

Prepare the sulfhydryl column by washing with a disulfide reducing agent. Apply 10 ml of freshly made lOmM DTT solution (15.4mg of DTT dissolved in 10ml of equilibration buffer). This treatment converts the immobilized ligands into a fully reduced form (free —SH groups). 

Even under the most inert atmosphere conditions, the 31CP/MAS spectrum of the immobilized ligand showed a major signal at 6 = 42 ppm (wrt 85% H3POO characteristic of phosphine oxide rather than phosphine. This could be quantitatively reduced by HSiCl3 and this surface reaction monitored by NMR but the subsequent exchange reaction (equation [5]) generated substantial quantities of phosphine oxide and a number of different isomeric complexes were f ormed. 

The signal is measured in terms of resonance units (RU) and the mathematical treatment is as described above. An increase in RU signal is proportional to the concentration of complex AB. The maximum of the specific change in signal then corresponds to a complete saturation of the immobilized ligand. 

The immobilized ligand is an essential factor that determines the success of an affinity chromatographic method [12]. The method which is used for affinity ligand immobilization is important because actual or apparent activity of the final column can be affected. Decrease in... 

The immobilized ligand to be studied is treated with regeneration buffer and then equilibrated in equilibration buffer. 

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