Dear all,

I am using Dde-Azide Agarose (https://clickchemistrytools.com/product/dde-azide-agarose/) and I am trying to click alkyne-tagged proteins via CuAAC to them (ON on 4C), wash, and elute with 2% Hydrazine.

However, when I analyse my elution via Coomassie or Silver Staining on a SDS-PAGE

I see only smear and no clear bands. I tried adjusting the pH of the 2% Hydrazine elution solution and EtOH precipitation of my eluted proteins followed by resuspension in 1xSample buffer and analysis on a SDS-PAGE. However, this did not help much.

I started wondering whether the Hydrazine does something to my proteins which makes them run differently or I can somehow not see them anymore?

Another option is, that I am using the wrong Hydrazine for elution? Currently I am using the 80% Hydrazine hydrate solution from Sigma (https://www.sigmaaldrich.com/catalog/product/sial/53860?lang=de®ion=DE) diluted to 2% and incubate the beads usually for 1hr.

Thanks already to all of you for your suggestions.