Accurately measure biotinylation levels with a new highly sensitive fluorescent assay.

The Thermo Scientific Fluorescence Biotin Quantitation Kit requires only 10 μl of sample to accurately measure the biotinylation level of biomolecules. This microplate-based assay is easy to perform by adding the Thermo Scientific DyLight Reporter to the biotinylated samples and diluted biocytin standards. The avidin fluoresces when the weakly interacting HABA (4'-hydroxyazobenzene-2-carboxylic acid) is displaced by the biotin. The amount of biotin is determined by comparing the sample's fluorescence to the biocytin standard curve. This assay requires half the sample volume and is much more sensitive than the microplate colorimetric HABA assay.

About the Fluorescence Biotin Quantitation Kit

The highly specific interaction of avidin with biotin is useful for designing nonradioactive purification and detection systems. Quantifying the degree of biotinylation is necessary for determining if biotinylation was successful and the amount of biotinylated molecule to use for a specific application. The Fluorescence Biotin Quantitation Kit contains a premix of fluorescent avidin with HABA and a biocytin standard. This fluorescent method is highly sensitive and accurate and requires only 10 μl of sample, whereas the traditional colorimetric method requires 100 μl of sample and Supplier I's fluorescent assay requires 50 μl of sample. Additionally, our kit more accurately quantitates biotin than Supplier I's fluorescent assay.

HABA (4'-hydroxyazobenzene-2-carboxylic acid) is a dye that weakly interacts with avidin and is commonly used in a colorimetric assay to quickly estimate the biotin-to-protein ratio; however, our new fluorescent method is more sensitive and accurate. The premix fluorescent avidin with HABA (DyLight Reporter) is added to the solution containing the biotinylated sample. Because of its higher affinity for avidin, biotin displaces the HABA, allowing the avidin to fluoresce. The amount of biotin is measured in a microplate by comparing the fluorescence to a biocytin standard curve.

Highlights for the Fluorescent Biotin Quantitation Kit:

• Fast – requires only 5 minute incubation
• Economical – requires only 10 μl of valuable samples
• Sensitive – linear working range of 10-60 picomoles of biotin (down to 750 ng of biotinylated IgG)

Comparison of results obtained using Thermo Scientific Fluorescent Biotin Quantitation Kit to those generated using a similar fluorescent kit from Supplier I. Rabbit and Goat anti-Rabbit IgG samples were spiked with a 2 fold molar excess of Sulfo-NHS-LC-Biotin (Product # 21327) and assayed for the amount of biotin according to the manufacturer's 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. The Thermo Scientific Fluorescent Biotin Quantitation Kit provided the most accurate estimation of biotin in each sample.

The working linear range of the Thermo Scientific Fluorescent Biotin Quantitation Kit is 5 to 60 picomoles. The standard curve was prepared using standards of 5 - 80 picomoles biocytin/10 μl. Ten microliters of each standard was pipetted into a microplate, in triplicate, and 90 μl of detection reagent was added. The mixtures were incubated for 5 minutes at room temperature and assayed for fluorescence using excitation 494 nm and emission 520 nm. The working range of the assay is 5 - 60 picomoles.

Related Products and Links:
HABA Reagent
Pierce Biotin Quantitation Kit – colorimetric method