Thermo Scientific Pierce HABA is 4'-hydroxyazobenzene-2-carboxylic acid, a simple reagent that enables spectrophotometric (colorimetric) estimation of biotinylation levels of labeled proteins and other molecules.
Determine the molar ratio of biotin incorporated into a protein using the HABA-Avidin method. The HABA dye (4'-hydroxyazobenzene-2-carboxylic acid ) binds to avidin to produce a yellow-orange colored complex which absorbs at 500nm. Free biotin will displace the HABA dye and cause the absorbance to decrease. A standard curve can be established using the free biotin to estimate the number of moles of biotin incorporated after biotinylating a protein.
- 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.Savage, M.D., Mattson, G., Desai, S., Nielander, G.W., Morgensen, S., and Conklin, E.J. (1992). Avidin-Biotin Chemistry: A Handbook. Rockford, IL: Pierce Chemical Co. (out of print)
- Janolino, V.G. et al. (1996). A spectrophotometric assay for biotin-binding sites of immobilized avidin. App. Biochem. and Biotech. 56, 1-7.
Review of Biotinylation
Online HABA Calculator – performs the calculations needed to determine biotin-protein ratios
Pierce Biotin Quantitation Kit – complete kit based on HABA dye
Fluorescence Biotin Quantitation Kit – more sensitive and accurate method