For easy, colorimetric detection and measurement of primary amines.
Trinitrobenzene sulfonic acid (TNBSA) reacts readily with the primary amino groups of amino acids in aqueous solution at pH 8 to form yellow adducts. No colored derivatives are formed with the secondary amino acids praline and hydroxyproline. The colored derivatives are monitored at 335-345 nm and have extinction coefficients in the range of 10,000-15,000.
TNBSA has been used as a hydrophilic modifying reagent for the detection of primary amines in samples containing amino acids, peptides or proteins. It is an excellent reagent for rapid qualitative and quantitative estimation of these biomolecules.
Product Detail
Highlights:
CAS#: 2508-19-2
Name: 2,4,6-trinitrobenzenesulfonic acid (TNBSA)
Alternative names: picrylsulfonic acid, trinitrophenylsulfonic acid, trinitrobenzene sulfonate (TNBS)
Couples with primary amines at pH 8, forming measurable yellow product (335-345 nm)
Supplied as 5% solution (w/v) of TNBSA in methanol, which is easily diluted approximately 1000-fold in assay buffer for typical protein and peptide applications
Applicable to solution or solid phase analysis
Example procedure for measuring primary amines in proteins (Hermanson, 2008, p. 127-128): 1.Dissolve or dialyze protein (20-200 µg/ml) in 0.1 M sodium bicarbonate buffer (pH 8.5). Avoid Tris or other amine-containing buffers. 2.Dilute supplied 5% TNBSA solution 500-fold in 0.1 M sodium bicarbonate buffer (pH 8.5). 3.Add 0.5 ml diluted TNBSA solution to 1 ml of protein solution. Mix well. 4.Incubate at 37°C for 2 hours. 5.Add 0.5 ml of 10% SDS and 0.25 ml of 1 N HCl to each sample to stop and stablize reaction. 6.Measure absorbance of solution at 335 nm. Determine concentration of primary amines by calculation from the extinction coefficient or by comparison to amino acid standards.
References: 1.Hermanson, G.T. (2008). Bioconjugate Techniques, Academic Press (Product # 20036) 2.Goodwin, J.F. and Choi, S-Y. (1970) Quantification of protein solutions with trinitrobenzenesulfonic acid. Clinical Chemistry. 16, 24-31. 3.Snyder, S.L. and Sobocinski, P.Z. (1975) An improved 2,4,6-trinitrobenzenesulfonic acid method for the determination of amines. Anal Biochem. 64, 284-288. 4.Drozdovskaya, N.R., et al. (1982) FEBS Lett. 150, 385. 5.Takahashi, S., et al. (1984) Chem. Lett. (Jpn). 1, 127.
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