Hi guys,
I'm currently working on the modification of Lys residues and need a quantitative assay to proof the content of free NH2 afterwards.
Is there a good protocol with TNBSA (2,4,6-trinitrobenzene sulfonic acid)?
Or do you use another agent? Because some literature says TNBSA is highly specific for primary amino groups (Lys + N-terminus), but other paper tell you that it will also react with e.g. Cys... Does anybody know?
Thanks for your help =)
Ina
Hi Ina,
More convenient for UV detection is the reaction of primary amines with phtaldialdehyde in 0.1 M borax and in the presence of mercaptoethanol. It is a very quick reaction (seconds) and the product absorbs at 340 nm, where the reagent itself has much weaker absorbance. SigmaAldrich sells the reagent ready for use. 1 mM reagent with 0.01 - 0.1 mM amine gives absorbance in ca.0.07 - 0.7 range. Besides, the product is fluorescent. Literature: V.K. Svedas, I.J.Galaev, I.L.Borisov, I.V. Berezin. The interaction of amino acids with o-phtaldialdehyde: A kinetic study and spectrophotometric assay of the reaction product. Anal. Biochem. 101 (1980) 188, plus many other papers.
Good luck !
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