I want to conjugate a hapten via its primary amine to carboxyl groups on a carrier protein using EDC/NHS. I understand this is normally done by first activating the carboxyl-containing molecule with EDC/NHS then adding the amine-containing molecule. To avoid carrier-carrier conjugation (as it is a protein with both amine and carboxyl groups), is it possible to first activate the hapten (which has an amine but no carboxyl groups) then add it to the carrier? I seem to remember an old paper saying this is possible but cannot find a recent protocol.
Using EDC/NHS chemistry no. Try adding EDC/NHS to a protein solution and you will see that crosslinking is difficult to happen. Hydrolysis is always the enemy. If you "activate" the carboxylic group with NHS for let's say 1 h, by the time you add the amine-containing molecule, the excess of EDC is already hydrolyzed if the reaction is occurring in aqueous medium. EDC/NHS chemistry sucks, I would activate the carboxylic groups and rapidly add the amine-containing molecules and pray for the desired reaction to occur.
I would use an extremely high concentration of hapten, added soon after activation or even at the same time. If the concentration is high enough, you should obtain hapten-protein conjugation rather than protein-protein. However, I do agree with Omar Gonzalez-Ortega, if you actually wanted to crosslink the proteins, you would not find it easy with EDC.
The most traditional way is to convert the amino hapten into a carboxylic acid by succinylation (with succinic acid anhydride). The product is isolated and subsequently activated with dicyclohexyl carbodiimide/NHS in a dry solvent (e.g. dioxane or conjugation grade dimethylformamide). After about 1 hr, any precipitate should be removed by centrifugation (solution needs to stay clear). The NHS ester can be isolated (best option) or used as a crude mixture. The protein is dissolved in a slightly basic buffer (pH 7.5-8.5, carbonate or borate) and the NHS ester solution is added slowly under stirring. Finally, the conjugate is centrifuged and purified e.g. on a Sephadex column.
Janis
I will think, primary NH2 is already an active/reactive group, if it is under basic condition.
Thank you all very much for your advices. I will give Omar Gonzalez-Ortega and Nick Gee suggestions a try, by increasing the amount of hapten and add it immediately after activation.
Why do you want to link your amine- containing hapten to a protein using the EDC/NHS system? What is wrong with using amine-amine cross-linking reagents? These are the usual and easy reagents for linking amine-containing haptens to proteins
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