Amplite® Colorimetric Biotin Quantitation Kit is designed to quantify the number of biotins attached to a protein or other macromolecule with a Nanodrop Spectrometer. The kit uses HABA (4’-hydroxyazobenzene-2-carboxylic acid) reagent that demonstrates a dramatic spectral change when bound to avidin. Biotin easily displaces HABA from the HABA/avidin complex, resulting in a decrease of absorption at 500 nm. By measuring the absorbance change before and after the addition of the biotin-containing sample at 500 nm, the amount of biotin in a sample can be quantified spectrophotometrically. The kit is best used to determine biotin concentration in the range from 2 to 16 µM. It is optimized to use with Nanodrop spectrometer. Biotin is a relatively small molecule that is routinely conjugated to antibodies and proteins with minimal interference of their biological activity. The avidin/streptavidin-biotin interaction is the strongest known binding pair between a protein and its ligand. The biotin-avidin interaction has been extensively explored for a variety of biological applications.