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AAT Bioquest

Amplite® Colorimetric Biotin Quantitation Kit

HABA Assay principle for quantifying biotinylation degree.
HABA Assay principle for quantifying biotinylation degree.
HABA Assay principle for quantifying biotinylation degree.
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Fax1-800-609-2943
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Certificate of OriginDownload PDF
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200
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OverviewpdfSDSpdfProtocol


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.

Platform


Absorbance microplate reader

Absorbance500 nm
Recommended plateClear bottom

Components


Example protocol


AT A GLANCE

Protocol summary
  1. Prepare HABA/Avidin assay mixture (180 µL)
  2. Add test samples (20 µL)
  3. Incubate at room temperature for 5 minutes
  4. Monitor absorbance decrease at 500 nm 

Important
Thaw all the kit components to room temperature before starting the experiment.

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

Avidin stock solution(100X)
Add 400 µL ddH2O into the vial of Avidin (Component A), Mix well.
Note     The unused 100X Avidin stock solution should be divided into single use aliquots and stored at -20 °C.

PREPARATION OF WORKING SOLUTION

HABA/Avidin assay solution
Add 200 µL of Avidin stock solution (100X) into 20 mL of HABA Assay Buffer (Component B), and mix them well.
Note     The unused portion of HABA/Avidin assay mixture might be stored at 4 °C up to one week.

SAMPLE EXPERIMENTAL PROTOCOL


Table 1.Layout of biotin-containing test samples, negative or positive controls in a white/clear bottom 96-well microplate
Note     NC= Negative Control, PC=Positive Control, TS=Test Samples.

NC NC TS TS
PC PC ... ...
TS TS ... ...
... ...   
... ...   
... ...   
... ...   
... ...   
Table 2. Reagent composition for each well
Positive ControlNegative ControlTest Sample
Compound C: 20 µLddH2O: 20 µL20 µL

Note     It is necessary to test the biotin-containing samples at several dilutions to ensure that the concentration of biotin is within the assay linear range, 2-16 μM of biotin (final concentration).
Note     Avoid buffers containing potassium, as it will cause precipitation in the assay.
Note     Free biotin must be separated from the biotinylated protein by gel filtration or dialysis.
  1. Add 20 µL each of biotin-containing samples, negative control (ddH2O or the same buffer used to dissolve biotin-containing sample), and positive Control (Component C) into a 96-well clear bottom microplate as described in Tables 1 and 2.
  2. Add 180 µL of HABA/Avidin assay solution into each well of the biotin-containing samples, negative control, and positive control to make the total biotin assay volume of 200 µL/well.
    Note     For a Cuvette Format, add 100 µL sample with 900 µL HABA/Avidin assay mixture.
  3. Incubate the reaction mixture at room temperature for 5 minutes by shaking on a plate shaker at 100-200 rpm, protected from light and avoid creating bubbles during pipetting.
  4. Monitor the absorbance decrease with an absorbance plate reader at 500 nm. 

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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
Fluorometric assay for quantitation of biotin covalently attached to proteins and nucleic acids
Authors: Batchelor RH, Sarkez A, Cox WG, Johnson I.
Journal: Biotechniques (2007): 503
Biotin-protein ratios and stability of biotinylated immunoglobulins as standards for the quantitation of biotin-binding immunoglobulins
Authors: Yazawa A, Fukuoka K, Honda H, Fukui T, Muratsugu M.
Journal: Biol Pharm Bull (2006): 1480
Dynamics of fluorescence dequenching of ostrich-quenched fluorescein biotin: a multifunctional quantitative assay for biotin
Authors: Wu Y, Simons PC, Lopez GP, Sklar LA, Bur and a T., undefined
Journal: Anal Biochem (2005): 221
A new chemically amplified electrochemical system for the detection of biological affinity reactions: direct and competitive biotin assay
Authors: Guo LH, Yang XQ.
Journal: Analyst (2005): 1027
Use of a sensitive EnVision +-based detection system for Western blotting: avoidance of streptavidin binding to endogenous biotin and biotin-containing proteins in kidney and other tissues
Authors: Banks RE, Craven RA, Harnden PA, Selby PJ.
Journal: Proteomics (2003): 558