Purified GTTR conjugate was produced as previously described (14). Briefly, an excess of gentamicin in 0.1 M potassium carbonate solution (pH 10) was mixed with TR succinimidyl esters (Invitrogen, CA) to minimize the possibility of over-labeling individual gentamicin molecules with more than one TR molecule and to preserve the polycationic nature of the conjugate, as previously described. After conjugation, reversed-phase chromatography, using C-18 columns (Grace Division, Discovery Science, IL), was used to purify the conjugate GTTR from unconjugated aminoglycosides and potential contamination by unreacted TR (27). The purified GTTR conjugate was aliquoted, lyophilized, and stored desiccated in the dark at −20°C until required.

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