This paper explores the conjugation of proteins with a range of different pI values to a flat surface http://pubs.acs.org/doi/abs/10.1021/ac980959t. They found that buffering the pH to about 1 pH point below the pI of the proteins helped to bind the primary amine of the protein to the carboxylic acid on the surface when activated with EDC NHS, but you are trying to do the reaction the other way around. The thermo scientific website info on EDC/NHS is also very useful http://www.piercenet.com/method/carbodiimide-crosslinker-chemistry and has details of the chemistry and how altering pH affects the rate and efficiency of the reaction. You may find that binding the proteins using EDC/NHS still causes the proteins to denature on the particles, even if you use the appropriate buffer, as it is difficult to control which groups on the protein bind to the particles, and you may get the proteins crosslinking in the solution rather than binding to the particles. You might just have to try it at a range of pH points and see which works the best. Good luck.

Hi, I did use EDC (not the NHS) at physiologic pH and room temperature and it worked (crosslinking was done). With this I'm not saying that I worked in the best conditions (e.g. as mentioned I didn't use the NHS) for the crosslinking efficacy but EDC can work even in non acid conditions.

Hi, I did use EDC (not the NHS) to conjugated an amine PEG to magnetic nanoparticles coated with DMSA (COOH groups). The reaction took place at room temperature, but pH has to be adjusted at 6. The molar ratio of COOH groups/amine groups/EDC was 1:1.5:1. The total amount of EDC was divided into five aliquots, one of which was added every 2 h, and the fifth 12 h after the previous addition (to avoid hydrolysis and EDC deactivation). However, the nanoparticles are not denatured after 12 hours of reaction at pH 6. Maybe you have to modify some experimental conditions for protein work. Good luck.

we did EDC-S-NHS in amine free buffer for functionalizing nanoparticles. We did the activation of carboxylate group using EDC/S-NHS at pH 5-6. Then we raised the pH to around 7 to 7.4 to link it to proteins. As the pH becomes basic the kinetics of the hydrolysis of the S-NHS activated -COOH group increases. WE had to raise the pH to betwenn 7-8 to avoid protein denanturation.2

Thank you for comments.

We did binding on proteins on nanomagnetic particles without loosing much on their efficiency.. Following papers describe the findings.

1. Biotechnol. Techniques 11 (1997) 493.

2. Immobilization of proteins and enzymes to fine magnetic particles, J,Magn. Magn. Matr. 201(1999)427.

Yes, you can do EDC-NHS at RT in PBS or at physiological pH. It works, efficency of conjugation can vary because of this (and many other factors too) but you can test it if you have access to an SPR or a QCM. At any rate if you're functionalizing nanoparticles you don't need to have a separate activation step, you can just do a one-pot functionalization, adding your EDC-NHS and amine-carrying conjugate in the desired proportions. This holds true if your conjugate does not carry any carboxyl groups.

The point is the IP of your protein, you should work at least one pH point bellow it

In our lab, we use EDC/Sulfo-NHS to couple COOH and NH2 groups in PBS buffer at room temperatures, and the pH value of buffer depends on the PI of protein, but not larger than 8.0

I am using EDC/s-NHS but it seems that cross linking is stronger than binding to the surface as after the first washing the Ab is gone ... I tried GSH to improve attachment to the surface but still no luck .... Can anyone help?

Thank you Maxwell. However, we are specifically targeting the carboxylic acid residues of protein.

i want to bind mannose to PLGA-COOH terminated by EDC and sulfo-NHS. but i confused a bout quantity of EDC and NHS in acidic PH. also How is the order of adding of material? TNX

I want to conjugate 6-aminocaproic acid ( NH2-(CH2)5COOH) to an aminated nanoparticle using EDC-NHS coupling. Please any information that can assist to do this without intermolecular recation between the NH2 and COOH of amino caproic acid

Derivatizing protein COOH with aminated ligand is tricky because the activated COOH will tend to react with protein amines. I have successfully done the conjugation that you are attempting at neutral pH by protecting the protein amines first. Would you mind share the reports that you mentioned?

hi mietta

please share you report or send by mail

[email protected]

thank you

Hello Yousef,

I am going to submit a paper on this conjugation method very soon. Once the paper is submitted I'll be happy to share. In the meantime would you like me to recommend you as a reviewer?

Also if there is anyone one else interested in reviewing this paper please let me know.

Thanks,

Mietta

Hi Mietta

Please share your report or send me through the following mail:

[email protected]

Thanks for your guidance

@ Amalia Ruiz Estrada is the process effective for AuNPs. Thank you very much