I thought that Tris is a primary amine, yet the protocols suggest using Tris to buffer the labelling reaction. Wouldn the Tris compete for the available fluor?
You may use Tris base with 2-D DIGE labelling because although it is a primary amine, it is much less reactive than the epsilon primary amine of lysine. This is due to its structure and the arrangement of its functional groups (Fig 1). Fig 1. Structures of (a) Tris base and (b) lysine. The amine group of Tris is attached to a quaternary carbon atom. This carbon has three bulky groups attached to it that sterically interfere with nucleophilic attack by the amine group on other molecules (e.g. CyDye™ DIGE Fluor). In addition, the three hydroxyl groups are electron-withdrawing and so reduce electron density on the nitrogen lone pair, thereby reducing its reactivity. The epsilon primary amine group of lysine is sterically unhindered and is remote from any electron-withdrawing groups, making it a much more reactive nucleophile in comparison. The poor nucleophilicity of the Tris amine group, enables the use of Tris as a buffer without compromising the efficiency of CyDye DIGE Fluor minimal d
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