Thermo Scientific Pierce Biotin Quantitation Kit includes aliquots of HABA reagent and biotinylated protein standard for convenient colorimetric determination of biotinylation levels in labeled antibodies and other proteins.
HABA dye (4'-hydroxyazobenzene-2-carboxylic acid) is a reagent that enables quick estimation of the mole-to-mole ratio of biotin to protein in a solution. The Pierce Biotin Quantitation Kit contains a premix of HABA and avidin and a biotinylated horseradish peroxidase (HRP) positive control. The HABA-Avidin Premix is supplied in convenient Thermo Scientific No-Weigh Microtube packaging, which eliminates the difficulties associated with weighing small quantities of reagent.
- Colorimetric – requires a plate reader or spectrophotometer set to measure at 500nm
- Reliable – proven, well-characterized method for determination of biotinylation levels
- Convenient – kit provides the essential reagents and standards to perform the assay with ease
- Flexible – adaptable to spectrophotometer cuvettes or standard plate readers with 96-well microplates
- Options – calculate results directly from absorbance values based on extinction coefficients using the procedure outlined in the instructions or use our online HABA calculator
- Robust – supplied HABA-avidin complex can be used over a wide range of pH and salt concentrations
To quantitate biotinylation, a solution containing the biotinylated protein is added to a mixture of HABA and avidin. Because of its higher affinity for avidin, biotin displaces the HABA and the absorbance at 500nm decreases proportionately. By this method, an unknown amount of biotin present in a solution can be quantitated in a single cuvette by measuring the absorbance of the HABA-avidin solution before and after addition of the biotin-containing sample. The change in absorbance relates to the amount of biotin in the sample by the extinction coefficient of the HABA-avidin complex.
|Chemical basis of the HABA biotin assay. The Thermo Scientific Pierce Biotin Quantitation Kit provides the essential reagents in convenient quantities for performing this assay.
- Green, N.M. (1965). A spectrophotometric assay for avidin and biotin based on binding of dyes by avidin. Biochem. J. 94, 23c-24c.
- Green, N.M. (1970). Spectrophotometric determination of avidin and biotin. Methods in Enzymol., Vol XVIII, Academic Press: New York, p. 418.
Review of Biotinylation
Online HABA Calculator – performs the calculations needed to determine biotin-protein ratios
HABA Reagent – stand-alone HABA dye
Fluorescence Biotin Quantitation Kit – more sensitive and accurate method