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Portelite™ Rapid Fluorimetric Biotin Quantitation Kit *Optimized to Use with CytoCite™ and Qubit™ Fluorometers*
Example protocol
PREPARATION OF STANDARD SOLUTIONS
https://www.aatbio.com/tools/serial-dilution/5539
PREPARATION OF WORKING SOLUTION
To prepare the Biotinylite™ Green working solution, add 100 µL of Biotinylite™ Green (10X) (Component A) into 900 µL of Assay Buffer (Component C), and mix thoroughly.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Preparation of Biotin Standards for thin-wall PCR tubes (Working Range: 0.75-30 µM in 20 µL).
Vial | Assay Buffer (µL) | Biotin Standard (30 µM) | Final Biotin Conc. in 20 µL (µM) |
STD7 | 0 | 20 | 30 |
STD6 | 4 | 16 | 24 |
STD5 | 12 | 8 | 12 |
STD4 | 16 | 4 | 6 |
STD3 | 18 | 2 | 3 |
STD2 | 19 | 1 | 1.5 |
STD1 | 19.5 | 0.5 | 0.75 |
BL | 20 | 0 | 0 |
Table 2. Preparation of Biotin Sample for thin-wall PCR tubes (Working Range: 0.75-30 µM in 20 µL).
Vial | Assay Buffer (µL) | Test Sample | Final Biotin Conc. in 20 µL (µM) |
TS | (20-V) µL | V µL | Dilute to 1 ~ 30 µM |
Note: Please estimate the concentration of biotin in your sample. Dilute it to achieve a concentration within the range of 1 to 30 µM. Adjust the volume to 20 µL using Assay Buffer (Component C).
Example: For a 1 mg/mL IgG-Biotin sample with a Biotin number of approximately 6.0, the Biotin concentration is around 40 µM. Therefore, you should use 5.0 µL of the sample and add 15.0 µL of Assay buffer (Component C) to achieve a final Biotin concentration of approximately 10 µM.
Dispense 180 µL of Biotinylite™ Green working solution into each tube.
Incubate the reaction at room temperature for 30 to 60 minutes.
Insert the samples into the CytoCite™ or Qubit® fluorimeters and monitor the fluorescence using the green fluorescent channel. For operational instructions for the CytoCite™, please refer to the following link:
https://devices.aatbio.com/documentation/user-manual-for-cytocite-fluorometer
On the Qubit® Home screen, select "Protein" and then choose "Read standards."
Insert each tube containing the standards into the sample chamber.
Close the lid and press the "Read Standards" button.
The instrument presents the measurements for each standard and sample.
Plot the standard curve, and use it to calculate the concentration of your samples.
References
Authors: Yu, Haibin and Liu, Dandan and Zhang, Yaowen and Tang, Ruijun and Fan, Xunan and Mao, Song and Lv, Lu and Chen, Fang and Qin, Hongtao and Zhang, Zhuohua and van Aalten, Daan M F and Yang, Bing and Yuan, Kai
Journal: eLife (2024)
Authors: Gautam, Bhaskarchand and Luo, Chun-Hao and Gao, Hua-De and Hsiao, Jye-Chian and Tseng, Hsian-Rong and Lee, Hsien-Ming and Yu, Hsiao-Hua
Journal: ACS applied bio materials (2023): 1611-1620
Authors: Tripathi, Ravi and Guglani, Anchala and Ghorpade, Rujuta and Wang, Binghe
Journal: Journal of enzyme inhibition and medicinal chemistry (2023): 2276663
Authors: Wang, Zhiming and Tan, Jing and Li, Mengxuan and Gao, Can and Li, Wenwen and Xu, Jing and Guo, Changchuan and Chen, Zhenzhen and Cai, Rong
Journal: Analytical chemistry (2023): 17533-17540
Authors: Nasrin, Fahmida and Khoris, Indra Memdi and Chowdhury, Ankan Dutta and Muttaqein, Sjakurrizal El and Park, Enoch Y
Journal: Biotechnology journal (2023): e2300125
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