Thermo Scientific Pierce Fluorescence Biotin Quantitation Kit requires only ten microliters of sample to accurately measure the biotinylation level of labeled antibodies and other biomolecules.
This microplate-based biotin assay is easy to perform by adding the supplied fluorescent 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 must less sample volume than the microplate colorimetric HABA assay and is much more sensitive.
Highlights:
- Fast – requires only 5 minute incubation
- Economical – requires only 10μL of sample (approx. 750ng of biotinylated IgG)
- Sensitive – linear working range of 10 to 60 picomoles of biotin
Includes:
Assay buffer, HABA-avidin fluorescent reporter solution, and biocytin control
Requires:
Black opaque 96-well microplate and a fluorescence plate reader (excitation 494nm; emission 520nm)
Product Details:
The highly specific interaction of avidin or streptavidin with biotin is the basis for many purification and detection systems. Quantifying the degree of biotinylation is necessary for assessing if a biotin-labeling procedure was successful and what amount of biotinylated molecule to use for a specific application.
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. The Fluorescence Biotin Quantitation Kit also uses HABA dye but is more sensitive and accurate. A premix of 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.
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| Chemical basis of the fluorescent biotin assay. Fluorescence (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). |
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| Assay standard curve and range of the Thermo Scientific Fluorescence Biotin Quantitation Kit. Biotin quantitation standards (biocytin at 0 to 100pmol per 10μL) were assayed in triplicate in a microplate upon addition of 90µL of detection reagent and incubation for 5 minutes at room temperature. Fluorescence was measured using excitation 494nm and emission 520nm. The results demonstrate that he linear working range of the assay is 5 to 60pmol per 10μL sample. |
The Fluorescence Biotin Quantitation Kit is requires only 10µL of sample. By contrast, the traditional colorimetric method requires 100µL of sample, and another leading supplier's fluorescent assay kit requires 50µL of sample. Our kit is more accurate than the other supplier's fluorescent biotin assay (Brand I).
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| Accuracy of fluorescent biotin assay kits. The 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. |
Related Products:
HABA Reagent
Pierce Biotin Quantitation Kit – colorimetric method
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