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Recently I conducted an experiment to conjugate amine-modified oligonucleotides with hydrogel particles having carboxyl groups. However, the fluorescence (from hybridized FAM-DNA) only appears on a small area of the particle and its shape is this ill-defined, like in the image below. Particle+FAM-DNA

My advisor said that "DNA is negative charge and carboxyl group also have negative charge so there is no possibility of conjugation, conjugation of streptavidin to particle, attaching biotinylated DNA and proceed with hybridization".

However, conjugation using NHS / EDC requires both an amine group and a carboxyl group. Does streptavidin have the requisite chemical groups to do such a conjugation? Streptavidin is Sigma Aldrich's 85878 (Streptavidin of Streptomyces avidinii, affinity purified, 10 mM potassium phosphate, lyophilized product of ≧ 13 U / mg protein).

PS: Please tell me if there is a secret to make it conjugate evenly on the surface.

PPS: We use 5 μM anime modified oligonucleotide and 100 nM FAM-DNA.

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  • $\begingroup$ I really don't get why this question had a downvote. $\endgroup$ – Roni Saiba Sep 30 '17 at 4:23
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The EDC-activated carboxylate is actually positively charged, though I cannot speak to the density of carboxylates in hydrogel or the extent of activation with your protocol.

It's not entirely clear from your question that you understand the mechanism of EDC activation, so you can read about it here (ThermoFisher).

enter image description here

Streptavidin has plenty of primary amines (lysine and N-terminus) to react with the EDC- or EDC/NHS-activated hydrogel, so it should conjugate readily. As it also has carboxylate groups, be sure to activate the resin first and then conjugate streptavidin to avoid unnecessary side-reactions.

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