I'm trying to perform EDC/NHS cross linking between 16-mercaptohexanoic acid (MHA) and proteins on the surface of gold nanoparticles. The gold nanoparticles are attached to a glass substrate through aminopropyltrimethsoxysilane, which binds gold through the amine-gold bond.
Originally, we tried quenching with ethanolamine, but our sensor went bonkers, which I thought might be the ethanolamine eluting AuNPs (ie competing for that amine on the crosslinker) but I'm not sure. I've read that Tris, Lysine, Glycine, hydroxlamine etc., are all decent alternatives. Does anyone here have any advice as to which, if any of these, is preferable for reasons that are surely over my head?
Thanks Kamyar.
I'm indeed trying to find the best quenching agent. For whatever reason, ethanolamine was pretty bad on our system. I'm hoping that Tris or glycine work better, but wondered if there's a preference. Like does tris have an advantage over glycine for example?
Hi Adam,
you might want to check out the the instructions for EDC and NHS at the ThermoFisher/Pierce website. There you can read, that all the typical chemicals used for quenching will result in modified carboxyls in your target. The best way to avoid this is probably raising the pH to 8.6 or higher, as NHS esters have a half life of only 10 minutes here before they hydrolyse and release the unmodified carboxyl group again.
What's the composition of glycine stop buffer that would be used for quenching?
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