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Preparing solutions for immobilization

For most immobilization approaches, the surface is activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Reagent solutions should be freshly prepared and mixed shortly before use. The efficiency of immobilization will be reduced if the solutions are not fresh.

Many protein ligands exhibit limited stability in the low ionic strength, low pH buffers typically used for covalent immobilization, and ligand solutions should be prepared in immobilization buffer as shortly as possible before use. If ligand is diluted into immobilization buffer from a stock solution, make sure the dilution factor is sufficiently high so that the ionic strength and pH of the immobilization buffer are not significantly affected. Rapid buffer exchange using desalting columns (e.g. NAP-10 from Cytiva or microspin techniques) is often a preferable alternative to dilution from stock solutions.

See also

List of help files

Covalent immobilization

Procedure for immobilization pH scouting

Pre-concentration

Immobilization buffer

Ligand immobilization levels

Ligand concentration