Amplite® Colorimetric Biotin Quantitation Kit

Amplite® Colorimetric Biotin Quantitation Kit is a colorimetric assay kit for quantifying biotin in solution or estimating the biotin-to-protein molar ratio in biotin-protein conjugates.

Colorimetric detection: Biotin displacement of HABA from avidin decreases absorbance at 500 nm.
Quantitation range: Measures biotin concentrations from 2 to 16 µM.
Specific binding chemistry: Uses the high-affinity avidin-biotin interaction for biotin quantitation.
Flexible format: Compatible with cuvette or microplate measurements at 500 nm.
Applications: Suitable for free biotin quantitation and determining biotinylation degree of protein conjugates.

HABA Assay principle for quantifying biotinylation degree.

Protocol

SDS

Catalog	Size	Price	Quantity
5522	200 Tests	Price

Overview

The avidin/streptavidin-biotin interaction is the strongest known non-covalent biological interaction (Kd = 10-15 M-1) between a protein and its ligand. One avidin binds four biotins. The bond formation between biotin and avidin/streptavidin is very rapid and, once formed, is unaffected by pH, organic solvents and other denaturing agents. Both avidin and streptavidin have essentially irreversible biotin-binding properties since bound biotin can only be released by denaturing the subunits of the proteins. The tight and specific binding of biotin and its derivatives to various avidins has been extensively explored for a number of biological applications. Amplite® Colorimetric Biotin Quantitation Kit provides a convenient method for estimating the molar ratio of biotin to protein in biotin-protein conjugates or for quantitating biotin concentration in a solution. The assay employs HABA (4'-hydroxyazobenzene-2-carboxylic acid), a reagent that shows dramatic spectral changes when bound to avidin. Biotin easily displaces HABA from the HABA/Avidin complex, resulting in a decrease of absorption at 500 nm. The kit is best used to determine biotin concentration in the range from 2 to 16 µM. The assay can be performed in a cuvette or microplate format.

Citations

View all 2 citations: Citation Explorer

Generation of shark Single-Domain antibodies as an aid for Cryo-EM structure determination of membrane Proteins: Use hyaluronan synthase as an example

Authors:

Deng, Penghui and Zhang, Xiaoyue and Wen, Jianqing and Xu, Mingce and Li, Pengwei and Wang, Hao and Bi, Yunchen

Journal:

Journal of Structural Biology: X (2025): 100126

Development of potential strategies for DNA quantification and DNA probes for detection of Legionella pneumophila

Authors:

Dulin, Parker James

References

View all 53 references: Citation Explorer

Detection of protein carbonyls by means of biotin hydrazide-streptavidin affinity methods

Authors:

Hensley K., undefined

Journal:

Methods Mol Biol (2009): 457

Red cell volume can be accurately determined in sheep using a nonradioactive biotin label

Authors:

Mock DM, Mock NI, Lankford GL, Burmeister LF, Strauss RG, Widness JA.

Journal:

Pediatr Res (2008): 528

Quantitation of biotin-binding immunoglobulins G, A, and M in Human Sera Using F(ab')2anti-human immunoglobulin-coated microplates

Authors:

Muratsugu M, Yazawa A, Fujiwara S, Nishida S, Fukui T.

Journal:

Biol Pharm Bull (2008): 507

Detection and quantitation of the activity of DNA methyltransferases using a biotin/avidin microplate assay

Authors:

Liebert K, Jeltsch A.

Journal:

Methods Mol Biol (2008): 149

Using liposomal fluorescent biolabels to develop an immunoaffinity chromatographic biosensing system for biotin

Authors:

Ho JA, Hung CH.

Journal:

Anal Chem (2008): 6405

Usage and storage

Intended use

Research Use Only (RUO)

Instrument settings

Absorbance microplate reader
Absorbance	500 nm
Recommended plate	Clear bottom

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Page updated on October 6, 2026

Component A: Avidin	1 vial
Component B: HABA Assay Buffer	1 bottle (40 mL)
Component C: d-biotin	1 vial (200 μL, 100 μM)