Ni-NTA-Cy3
(Ni2+-NTA)2-Cy3 Synthesis Ni2+-loaded, bis-NTA-Cy3 ((Ni2+-NTA)2-Cy3) was synthesized using a modification of the method described in [17]. Nα,Nα-bis (carboxymethyl)-L-lysine hydrate (Sigma, St. Louis, MO) (26 mg, 100 μmol) was dissolved in 1.6 ml 0.1 M Na2CO3 (pH 11) and added to 1 vial of Cy3 bis-Reactive Dye (200 nmol, GE Healthcare, Piscataway, NJ). After 1 h at room temperature (RT) in the dark, the reaction mixture was passed through a Sep-Pak Plus C18 cartridge (Waters, Milford, MA) and the bound material was eluted with 60% methanol and dried. The mixture was dissolved in 10% methanol and separated on preparative thin layer chromatography plates (Uniplate Silica Gel G, Analtech, Newark, DE) using NH4OH:ethanol:water (33:21:6, v/v/v) as mobile phase. NTA2-Cy3 (Rf = 0.24) was eluted with H2O and dried. Its identity was confirmed by mass spectrometry (MALDI-TOF): m/z = 1205.51 (calculated 1205.46). Nickel ions were loaded into the NTA2-Cy3 as described [17] and the final product, (Ni2+-NTA)2-Cy3, was dissolved in H2O and stored at -80°C. The (Ni2+-NTA)2-Cy3 product had absorbance and fluorescence excitation maxima at 553 nm and an emission maximum at 563 nm, consistent with published values [17]. Dye concentrations were measured by absorbance at 550 nm (ε550 = 1.5 × 105 M-1 cm-1). The typical yield from the reaction was 24% relative to the starting Cy3 bis-Reactive Dye. (Ni2+-NTA)2-Cy5 has been made following this procedure [17] and the synthesis of other (Ni2+-NTA)2-Cy derivatives (i.e. Cy2, Cy5.5, Cy7) is not expected to require major modifications of this protocol.
Of note, there was a 2-fold increase in fluorescence intensity when (Ni2+-NTA)2-Cy3 was incubated with EDTA, suggesting that Ni2+ binding quenches fluorescence. More severe quenching was seen for the analogous (Ni2+-NTA)2-Cy5 compound (data not shown). Quenching by the coordinated Ni2+ has been reported for other Ni2+-NTA-conjugated fluorescent probes