NHS esters efficiently react with primary amines (R-NH2) of proteins and other amine-containing molecules. Biodyes BDP-NHS esters have been bound to bovine serum albumin (BSA) and antibodies by the following procedure:
- Combine 0.5 mg BSA/antibody in 0.05 mL of a phosphate buffer (pH = 7.0) with 10 microliters of a solution made from 0.2 mg of Biodye NHS ester in dry DMSO and incubate for 4 hours at room temperature.
- Labeled antibodies are typically separated from free BODIPY™ dye using a gel filtration column Zeba Spin Desalting Columns (ThermoFisher)
- Degree of labeling DOL is determined by UV/vis spectroscopy
DOL = Amax x e protein/Aprotein x e dye
Amax (dye) = 0.69
e (dye) = 70,000 M-1cm-1
Aprotein = 0.22
e (BSA) = 44,000 M-1cm-1
DOL(calculated) = 2.0
This indicates that 2 dye molecules bind to each BSA protein molecule.

Electronic spectroscopy of BSA-dye conjugate after filtration.