Coupling BG-GLA-NHS to Proteins
NHS coupling to proteins is often performed using an excess of NHS ester. Depending on the number of lysine residues in the protein, it may be necessary to stop the labeling reaction before it reaches completion to avoid damaging the protein or causing it to unfold. A typical coupling involves reacting the NHS ester with exposed primary amines on the surface of the proteins to form an amide bond. The reaction can be carried out at pH 7-9 (typically pH 8.3), temperatures from 4–37°C and incubation times ranging from 30 min to overnight. Care must be taken to avoid primary amine containing buffers such as Tris or ammonium ions.
Coupling BG-GLA-NHS to Amine-Containing Compounds
A typical procedure involves equimolar quantities of NHS ester, amine to be coupled, and triethylamine in DMF at 30°C overnight. The purification strategy will depend on the label used. Good results have been obtained with both HPLC and silica gel chromatography