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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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OverviewpdfSDSpdfProtocol


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.

Platform


Fluorescence microplate reader

Excitation490 nm
Emission530 nm
Cutoff515 nm
Recommended plateSolid black

Components


Example protocol


PREPARATION OF STANDARD SOLUTIONS

For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/5538

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).

PREPARATION OF WORKING SOLUTION

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.

SAMPLE EXPERIMENTAL PROTOCOL

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

STD7
STD7
TS1
TS1
STD6
STD6
TS2
TS2
STD5
STD5
...
...
STD4
STD4
...
...
STD3
STD3
...
...
STD2
STD2
...
...
STD1
STD1
...
...
BL
BL
...
...

 Table 2. Reagent composition for each well.

Well
Volume
Reagents
STD7~STD1
50 µL
Biotin Serial Dilution (0.094 - 6 µM)
BL
50 µL
Assay Buffer Control
TS
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.

Images


References


View all 50 references: Citation Explorer
Supported Immunohistochemical Staining of Mucins.
Authors: Higashi, Michiyo
Journal: Methods in molecular biology (Clifton, N.J.) (2024): 101-109
Modular Set of Reagents in Lateral Flow Immunoassay: Application for Antibiotic Neomycin Detection in Honey.
Authors: Barshevskaya, Lyubov V and Sotnikov, Dmitriy V and Zherdev, Anatoly V and Dzantiev, Boris B
Journal: Biosensors (2023)
A dual-labeled fluorescent probe for visualization of dextranase activity in a simulated food digestion system.
Authors: Sun, Rongjuan and Liu, Weiji and Kirk, Timothy V and Chen, Xiao Dong
Journal: Food chemistry (2023): 134744
Immunohistochemistry of Immune Cells and Cells Bound to in vivo Administered Antibodies in Liver, Lung, Pancreas, and Colon of B6/lpr Mice.
Authors: Adam, Kieran and Mor, Adam
Journal: Bio-protocol (2022)
A linear-polymer-based lactoferrin-selective recognition element for an ELISA mimic: A proof of concept.
Authors: Goicolea, M A and Gómez-Caballero, A and Saumell-Esnaola, M and García Del Caño, G and Unceta, N and Sallés, J and Barrio, R J
Journal: Analytica chimica acta (2022): 339309
Visualization of the binding between gintonin, a Panax ginseng-derived LPA receptor ligand, and the LPA receptor subtypes and transactivation of the EGF receptor.
Authors: Choi, Sun-Hye and Lee, Ra Mi and Cho, Han-Sung and Hwang, Sung Hee and Hwang, Hong-Ik and Rhim, Hyewhon and Kim, Hyoung-Chun and Kim, Do-Geun and Cho, Ik-Hyun and Nah, Seung-Yeol
Journal: Journal of ginseng research (2022): 348-356
Design and Synthesis of Neutralizable Fondaparinux.
Authors: Zhang, Liangwei and Liu, Yating and Xu, Zhuojia and Hao, Tianhui and Wang, Peng George and Zhao, Wei and Li, Tiehai
Journal: JACS Au (2022): 2791-2799
Synthesis of the matriglycan hexasaccharide, -3Xylα1-3GlcAβ1-trimer and its interaction with laminin.
Authors: Tamura, Takahiro and Omura, Yuka and Kotera, Kota and Ito, Ryota and Ohno, Shiho and Manabe, Noriyoshi and Yamaguchi, Yoshiki and Tamura, Jun-Ichi
Journal: Organic & biomolecular chemistry (2022): 8489-8500
Decreased Krüppel-like factor 4 in adenomyosis impairs decidualization by repressing autophagy in human endometrial stromal cells.
Authors: Mei, Jie and Sheng, Xiaoqiang and Yan, Yuan and Cai, Xinyu and Zhang, Chunxue and Tian, Jiao and Zhang, Mei and Zhou, Jidong and Shan, Huizhi and Huang, Chenyang
Journal: BMC molecular and cell biology (2022): 24
Synthesis and Antitumor Evaluation of Glutathione Responsive Self-Immolative Disulphide Linked Camptothecin-Biotin Conjugate.
Authors: Kaur, Amardeep and Dhiman, Shikha and Lee, Hong Boon and Sharma, Manu
Journal: Anti-cancer agents in medicinal chemistry (2022): 3182-3192