AF488 NHS ester is an amine-specific fluorescence probe (Em=525 nm) that reacts with sulfhydryl groups and amines in aqueous and biological samples, altering their chemical structure and fluorescence properties after derivatization [1].

AF488 NHS ester is used for fluorescent labeling of IgG and in screening anti-IgG solid-phase peptide libraries. To begin, lyophilized IgG is dissolved at a concentration of 5 g/L in a solution of 50 mM sodium phosphate and 20 mM sodium chloride at pH 8.3. Then, 1 mg of AF488 NHS ester is dissolved in 100 µL of extra dry DMF and added to 1 mL of the IgG solution, followed by room temperature incubation with rotation for 1 hour. The sample is collected using an Amicon Ultra 0.5-mL centrifugal filter unit equipped with a 3-kDa MWCO filter. Deprotected hexamer or tetramer libraries are washed three times in 50 mM sodium phosphate and 150 mM sodium chloride at pH 7.4 (PBS) using five times the volume of resin to equilibrate. IgG-AF488 is diluted in a solution of 50 mM sodium phosphate, 150 mM sodium chloride, and 0.2% Tween (pH 7.4) to a final concentration of 1.3 mg/mL. The solutions prepared in steps (4) and (5) are incubated overnight at 2-8°C. The resin beads are washed with 50 mM sodium phosphate, 150 mM sodium chloride, and 0.1% Tween 20 at pH 7.4 (PBS-T). Each resin bead is deposited in a 96-well plate with 40 µL of PBS-T per well, and imaging is conducted using a fluorescence microscope at 10x magnification. Alexa Fluor 488 fluorescence measurement and screening are performed with excitation at 480 nm and emission intensity threshold at 510 nm. The AF488 NHS ester storage solution is prepared at a concentration of 20 mM in DMF, with storage recommended in aliquots at -20°C, protected from light.