Amine Coupling: A ligand is immobilized onto slide with available carboxylate groups. The carboxylate groups on the surface of the slide are modified with linker, a mixture of EDC and NHS to form an ester. A covalent amide bond is then formed to the ligand which must have at least one free primary amine group. Injection of ethanolamine removes any loosely bound ligand. Analyte is injected over the surface at a range of concentrations higher and lower than the expected equilibrium dissociation constant (K).

Chemicals:

EDC (Sigma Aldrich Cat. No. 03449-5G)

NHS (Pierce Cat No. 24500) 10mM

Sodium acetate pH 5.2 (need pH about 2 units below pI of ligand to be coupled)

1M ethanolamine pH 8.5 (Sigma >98% Cat No. EO135 – you dilute to 1M and pH to 8.5 with HCl)

PBST (Sigma Cat No. P 3563) or whatever buffer is needed

Immobilisation:

Flowrate: 20-50 µL/min

SPR prism temperature: 25 °C

Steps:

Weigh 40 mg EDC and 10 mg NHS into a suitable vial and dilute with 0.9 mL of water. Dissolve and inject as quickly as possible after diluting. Inject over both channels for 7 minutes 20 µL/min.

Prepare a solution of 30-50 µg/mL of proteine in 10mM sodium acetate pH 5.2 buffer. Over the sample channel only, inject for about 10 minutes or until a maximum has been achieved.

Inject over both channels 1M ethanolamine HCl for 10 minutes (blocked the unspecific binding sites).

Inject the solution of the ligand (other protein) for both channel, than remove the excess amount by running buffer.