Chemical basis of the fluorescent biotin assayFluorescence (green stars) of the DyLight Reporter (fluor-tagged avidin) increases when HABA dye (H) is displaced by biotin (B) contributed by a known amount of biotinylated protein. Measurement is made in black opaque 96-well microplates with a fluorescence plate reader (excitation 494nm; emission 520nm).
Accuracy of fluorescent biotin assay kitsThe Thermo Scientific Fluorescence Biotin Quantitation Kit is more accurate than the kit from another leading supplier (Brand I). Rabbit and goat IgG samples were spiked with a 2-fold molar excess of Sulfo-NHS-LC-Biotin (Part No. 21327) and assayed for the amount of biotin according to manufacturers' instructions. A linear regression curve fit was applied to the linear portion of each standard curve and the resulting equation was used to calculate picomoles of biotin per sample. This value was divided by the picomoles of protein in the sample to generate the ratio of moles of biotin per mole of protein.
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