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Amplite® Fluorimetric Biotin Quantitation Kit

The Biotin Standard Curve was determined using a black bottom 96-well plate and the Amplite® Fluorimetric Biotin Quantitation Kit, with measurements taken on a fluorescence microplate reader at an Ex/Em = 490/530 nm and Cutoff = 515 nm.
The Biotin Standard Curve was determined using a black bottom 96-well plate and the Amplite® Fluorimetric Biotin Quantitation Kit, with measurements taken on a fluorescence microplate reader at an Ex/Em = 490/530 nm and Cutoff = 515 nm.
The Biotin Standard Curve was determined using a black bottom 96-well plate and the Amplite® Fluorimetric Biotin Quantitation Kit, with measurements taken on a fluorescence microplate reader at an Ex/Em = 490/530 nm and Cutoff = 515 nm.
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Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
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Amplite® Fluorimetric Biotin Quantitation Kit uses Biotinylite™ Green, a fluorogenic biotin sensor. Biotinylite™ Green is almost non-fluorescent and give strong green fluorescence upon interaction with a biotin or biotin conjugate. The concentration of biotin is proportional to the fluorescence intensity of Biotinylite™ Green. The amount of biotin is determined by comparing a sample’s fluorescence to the predetermined biotin standard curve. This fluorescence-based assay is much more sensitive than the commonly used colorimetric HABA assay. 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.


Fluorescence microplate reader

Excitation490 nm
Emission530 nm
Cutoff515 nm
Recommended plateSolid black


Example protocol


For convenience, use the Serial Dilution Planner:

Biotin Standard
To prepare a 6 µM Biotin Standard (STD7), add 10 µL of 300 µM Biotin Standard stock solution to 490 µL of Assay Buffer (Component C). Then, create 1:2 serial dilutions by taking 200 µL of this Biotin Standard solution and diluting it with equal volumes of Assay Buffer (Component C) to obtain serially diluted standards (STD7-STD1).


Biotinylite™ Green Working Solution
  1. To prepare the Biotinylite™ Green working solution, add 100 μL of Biotinylite™ Green (10X) (Component A) to 900 μL of Assay Buffer (Component C) and mix thoroughly.


Table 1. Layout of biotin standards and test samples in a solid black 96-well microplate.

STD = Biotin Standard (STD1-STD7, 0.094 ~ 6 µM), BL = Blank Control, TS = Test Samples


 Table 2. Reagent composition for each well.

50 µL
Biotin Serial Dilution (0.094 - 6 µM)
50 µL
Assay Buffer Control
50 µL
Test Sample
  1. Prepare the Biotin standard (STD1~STD7), blank controls (BL), and test samples (TS) as outlined in Tables 1 and 2, using 25 µL of reagent per well for a 384-well plate instead of 50 µL.

  2. Add 50 µL of Biotinylite™ Green working solution to each well containing Biotin standards (STD1~STD7), blank controls (BL), and test samples (TS), ensuring the total volume in each well reaches 100 µL. For a 384-well plate, use 25 µL of the working solution per well.

  3. Incubate the reaction at room temperature for 10 to 30 minutes, protected from light.

  4. Monitor the fluorescence increase at Ex/Em = 490/530 nm (Cutoff = 515 nm) with a fluorescence microplate reader.



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