Amplite? Colorimetric Biotin Quantitation Kit. The avidin/streptavidin-biotin interaction is the strongest known non-covalent biological interaction (Kd = 10-15 M-1) between a protein and its ligand. One avidin binds four biotins. The bond formation between biotin and avidin/streptavidin is very rapid and, once formed, is unaffected by pH, organic solvents and other denaturing agents. Both avidin and streptavidin have essentially irreversible biotin-binding properties since bound biotin can only be released by denaturing the subunits of the proteins. The tight and specific binding of biotin and its derivatives to various avidins has been extensively explored for a number of biological applications. Amplite? Colorimetric Biotin Quantitation Kit provides a convenient method for estimating the molar ratio of biotin to protein in biotin-protein conjugates or for quantitating biotin concentration in a solution. The assay employs HABA (4'-hydroxyazobenzene-2-carboxylic acid), a reagent that shows dramatic spectral changes when bound to avidin. Biotin easily displaces HABA from the HABA/Avidin complex, resulting in a decrease of absorption at 500 nm. The kit is best used to determine biotin concentration in the range from 2 to 16 μM. The assay can be performed in a cuvette or microplate format.