N3-C3-NHS ester

Park JY, et al. Theranostics, 2019, 9(12), 3398-3409.

N3-C3-NHS ester is used for the coupling reaction of albumin with Azadibenzocyclooctyne-NHS ester (ADIBO-NHS).
The basic steps are as follows:
Albumin is dissolved in PBS buffer at a concentration of 1 mg/mL and 0.5 mg/0.5 mL of the solution is added to each reaction vial.
ADIBO-NHS and N3-NHS are dissolved in DMSO at a 5 mg/125 μL concentration and added to PBS in a total volume of 500 μL.
The NHS compounds are added to the reaction vials at different molar ratios (NHS compounds:albumin, 1.1:1, 3.4:1, 5.7:1, 8.1:1, 11.4:1, 17.2:1, and 23:1, respectively).
The reaction vials are incubated for 2 h at room temperature and then analyzed using PBS as a mobile phase by size exclusion chromatography.
Droplets of each fraction are placed on a glass microfiber filter and stained with Caulmers Brilliant Blue. The grades stained blue are collected for albumin concentration analysis.
Albumin concentration is quantified by determining the absorbance ratio at 260/280 nm using UV-visible spectroscopy.
The albumin-ADIBO conjugate is lyophilized for further experiments.