Hydrazides are used for the labeling of molecules bearing carbonyl groups, such as aldehydes, and ketones.

Scheme for aldehyde-functionalized biomaterial labeling with hydrazide-based dyes*

Hydrazide-based labeling

Carbonyl groups can be easily generated from vicinal diol groups of glycoproteins by periodate oxidation - this provides a good method for the labeling of glycoproteins. In case of antibodies, this is especially useful because the glycosylation site is located far from the epitopes.

We did not place the protocol on our web site yet, but it looks as follows:

Omit steps 1-4 if an aldehyde group is already present in the molecule

1. If molecule does not contain carbonyl groups, but contains a sugar or another 1,2-diol, cleavage of diol is necessary. Prepare a solution of meta-periodate at 20 mM in 0.1M sodium acetate buffer, pH 5.5 This solution should be used immediately as prepared:

The reaction of sodium periodate with sugar residues produces aldehydes for further conjugation reactions*.  R and R' represent connecting sugar monomers of the polysaccharide. Red asterisks indicate sites of diol cleavage.

An example of sugar residues pre-modification for labeling with aldehyde-reactive dyes
2. Prepare the protein solution at 5 mg/mL in 0.1M sodium acetate buffer, pH 5.5.
3. Add 1 ml of periodate solution to 1 ml of protein solution. Mix, and incubate for 5 min.
4. Desalt or dialyze against 0.1 M sodium acetate pH 5.5.
5. Prepare hydrazide solution in DMSO (50 mM).
6. Add 200 μl of hydrazide solution to 2 mL of protein solution, and incubate for 2 h at RT.
7. Purify the labeled protein by gel filtration.

The recommended article to read about labeling with hydrazides is: Wilchek and Bayer, 1987.

*The images were taken from ThermoFisher webpage for Carbonyl-reactive Crosslinker Chemistry