I'd like to use EDC/NHS in an excess platform to conjugate an amine group to the carboxyl group of a polysaccharide. Considering this fact that i don't have the optimize mol ratio for my components i need to assume 2,3,5,...mol of EDC,NHS to the mol of one of these two components and i'm not sure which one would be the better choice.
I do not know your specific system. In general, you may use a significant excess of NHS (e.g. 2x of carboxyl) and an equimolar amount (e.g. 1x of carboxyl) of EDC. Too much EDC will lead to byproducts, particularly with the amino groups. Please note that EDC is extremely sensitive to hydrolysis. Any open vial might be inactive.
Thank you Stjepan Krešimir Kračun for your answer, totally useful
As you said the molecule carrying the amine group is really small in comparison with the polysaccharide and my calculation end up with a really small quantity of this molecule so I started to guess instead.
as I get , you suggested an overnight reaction including the reagent and the polysaccharide and later adding the amine molecule due to the by products problem, is that right?
Thank you Michael G. Weller for your answer
just wondering why is the mol ratio of NHS is different from EDC?
I have many references that these two reagents are used in the same mol ratio.
And is it possible to calculate them in comparison with the Amine group? (e.g. 5x Amine)
NHS can not do any harm to your molecules, therefore a higher access can be used and lead to a faster reaction and a higher yield, particularly, if you can not calculate the concentration of your carboxylic groups properly. It is not really advisable to calculate the reagent relative to the amino groups, because the reaction between the carboxylic group and EDC is always the first step (see lower link). Therefore, two-step protocols are usually preferable. In this case, you can also use a larger excess of amine in the second step, if it is not too expensive.
If you want to read more about bioconjugation, the book of Greg Hermanson can be highly recommended:
http://www.thermofisher.com/order/catalog/product/26166
https://www.thermofisher.com/de/de/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/carbodiimide-crosslinker-chemistry.html
We used a 1:1 molar ratio of EDC:NHS for our application but that was for large-scale cross-linking of two proteins: Article Acanthamoeba actin and profilin can be cross-linked between ...
- Badges
- Science topic
- Similar topics
- Chemistry
- Pharmacology
- Pharmaceuticals
- Drugs