Portelite™ Rapid Fluorimetric Biotin Quantitation Kit Optimized to Use with CytoCite™ and Qubit™ Fluorometers

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Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
International	See distributors
Bulk request	Inquire
Custom size	Inquire
Shipping	Standard overnight for United States, inquire for international

Physical properties

Solvent

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Freeze (< -15 °C); Minimize light exposure

Overview SDS Protocol

Portelite™ Rapid Fluorimetric Biotin Quantitation Kit is specifically optimized to quantify biotin and biotin conjugates with a portal fluorometer such as CytoCite™ and Qubit™ Fluorometer. The 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

Qubit Fluorometer

Excitation	490 nm
Emission	530 nm
Instrument specification(s)	Green Fluorescent Channel

Components

Example protocol

PREPARATION OF STANDARD SOLUTIONS

For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/5539

Biotin Standard

Add 10 µL of the 300 µM Biotin Standard stock solution to 90 µL of Assay Buffer (Component C) to create a 30 µM Biotin standard (STD7). Refer to Table 1 for detailed serial dilution instructions.

PREPARATION OF WORKING SOLUTION

Biotinylite™ Green 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.

Run Biotin Assay

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

Sample Protocol for Qubit® Fluorimeter

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.

Images

Figure 1. The Biotin Standard Curve was determined using the Portelite™ Rapid Fluorimetric Biotin Quantitation Kit, with measurements taken on a Qubit™ 4 Fluorometer using the blue (470 nm) channel.

References

View all 47 references: Citation Explorer

Tissue-specific O-GlcNAcylation profiling identifies substrates in translational machinery in Drosophila mushroom body contributing to olfactory learning.
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)

Nano-On-Nano: Responsive Nanosubstrate-Mediated Liposome Delivery with High Cellular Uptake Efficiency.
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

Biotin conjugates in targeted drug delivery: is it mediated by a biotin transporter, a yet to be identified receptor, or (an)other unknown mechanism(s)?
Authors: Tripathi, Ravi and Guglani, Anchala and Ghorpade, Rujuta and Wang, Binghe
Journal: Journal of enzyme inhibition and medicinal chemistry (2023): 2276663

Clickable Photoreactive ATP-Affinity Probe for Global Profiling of ATP-Binding Proteins.
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

Development of disposable electrode for the detection of mosquito-borne viruses.
Authors: Nasrin, Fahmida and Khoris, Indra Memdi and Chowdhury, Ankan Dutta and Muttaqein, Sjakurrizal El and Park, Enoch Y
Journal: Biotechnology journal (2023): e2300125

Red-Absorbing Ru(II) Polypyridyl Complexes with Biotin Targeting Spontaneously Assemble into Nanoparticles in Biological Media.
Authors: Vinck, Robin and Gandioso, Albert and Burckel, Pierre and Saubaméa, Bruno and Cariou, Kevin and Gasser, Gilles
Journal: Inorganic chemistry (2022): 13576-13585

Lipid-mediated biosynthetic labeling strategy for in vivo dynamic tracing of avian influenza virus infection.
Authors: Liu, Junfang and Su, Minhong and Chen, Xin and Li, Zhongli and Fang, Zekui and Yi, Li
Journal: Journal of biomaterials applications (2022): 1689-1699

Bead-Based Multiplexed Droplet Digital Polymerase Chain Reaction in a Single Tube Using Universal Sequences: An Ultrasensitive, Cross-Reaction-Free, and High-Throughput Strategy.
Authors: Gu, Zhejia and Sun, Tong and Guo, Qingsheng and Wang, Yao and Ge, Yunfei and Gu, Hongchen and Xu, Gaolian and Xu, Hong
Journal: ACS sensors (2022): 2759-2766

Spatiotemporal Proximity Labeling Tools to Track GlcNAc Sugar-Modified Functional Protein Hubs during Cellular Signaling.
Authors: Liu, Yimin and Nelson, Zachary M and Reda, Ali and Fehl, Charlie
Journal: ACS chemical biology (2022): 2153-2164

Targeted delivery of quercetin by biotinylated mixed micelles for non-small cell lung cancer treatment.
Authors: Li, Kangkang and Zang, Xinlong and Meng, Xiangjun and Li, Yanfeng and Xie, Yi and Chen, Xuehong
Journal: Drug delivery (2022): 970-985

Documents

Safety data sheet
Product protocol
Certificate of analysis

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Portelite™ Rapid Fluorimetric Biotin Quantitation Kit *Optimized to Use with CytoCite™ and Qubit™ Fluorometers*

OverviewSDSProtocol