6-Carboxytetramethylrhoda?mine (6-TAMRA) is used to create fluorescent DNA probes and labeled proteins/peptides. The corresponding succinimidyl ester is a single isomer fluorophore most commonly used in automated DNA sequencing.
1).Solvents:For the most part, reactive dyes are hydrophobic molecules and should be dissolved in anhydrous dimethylformamide (DMF) or dimethylsulfoxide (DMSO).
2).Reaction pH:The labeling reactions of amines with succinimidyl esters are strongly pH dependent. Amine-reactive reagents react with non-protonated aliphatic amine groups, including the terminal amines of proteins and the e-amino groups of lysines. Thus amine acylation reactions are usually carried out above pH 7.5. Protein modifications by succinimidyl esters can typically be done at pH 7.5-8.5, whereas isothiocyanates may require a pH 9.0-10.0 for optimal conjugations.
3).Reaction Buffers:Buffers that contain free amines such as Tris and glycine and thiol compounds must be avoided when using an amine-reactive reagent. Ammonium salts (such as ammonium sulfate and ammonium acetate) that are widely used for protein precipitation must also be removed (such as viadinlysis) before performing dye conjugations.
4).Reaction Temperature:Most conjugations are done at room temperature. However, either elevated or reduced temperature may be required for a particular labeling reaction.
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