EDC/NHS-coupling chemistry

Studies of the interaction of IL-6 and the sIL-6R were monitored by SPR detection using a BIAcore instrument [1] (Pharmacia, Uppsala). Immobilisation of the respective proteins to the carboxymethylated dextran matrix coating the gold sensor chip was performed using the EDC/NHS coupling chemistry as previously described for IL-6 and sIL-6R [3, 7, 8]. Regeneration of the sensor surface for IL-6 and sIL-6R was performed with lOmM HCl for 3 min [8] and 4M MgCl2 in lOmM Tris-HCl buffer, pH 7.4 for 1 min respectively. 

Soper et al. [211] presented the fabrication of DNA microarrays onto PMMA surfaces using a UV modification protocol as shown in Fig. 11. Briefly, the PMMA surface was first activated via exposure to UV irradiation, which produced carboxylic acid functional groups onto its surface. EDC/NHS coupling chemistry was then used to facilitate the formation of succinimidyl ester intermediates on the surface, which allowed for the covalent attachment of amine-terminated... 

For protein immobilization the most commonly used method is the EDC-NHS affinity ligand coupling chemistry as described for DNA bonding to glass substrates. An advanced surface coating method for protein microarrays was developed by the company MicroSurfaces. A polyiethylene glycol) (PEG) surface coating. 

The immobilization of anti-P-endorphin to the carboxymethylated dextran (which is on the surface of the resonant layer) was via NHS/EDC chemistry. Prior to antibody coupling, the carboxymethylated dextran was activated twice with 0.4 M EDC/0.1 M NHS for 10 min. Anti-P-endorphin was coupled twice to the dextran layer in 10 mM sodium acetate buffer, pH = 5.0, at 25 pg/ml to ensure maximum loading. After coupling, the free activated carboxyl group was blocked with 200 pi of 1 M ethanolamine for two minutes. Finally, the cuvette with immobilized anti-p-endorphin was washed twice with 20 mM HCl and twice with PBS/0.05% tween 20, to eliminate the non-covalently bound antibody. The binding of the peptides to the antibody was carried out in PBS, pH=7.4, at 25°C. After the baseline was established for 150 pi of PBS, 50 pi of 3 mg/ml (0.1 mg/ml/peptide) crude peptides in water was added to the cuvette and the binding was monitored. When equilibrium was achieved (approximately 10 min), the unbound peptides were flushed away. 

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