Comparison of Three Common Amine Reactive Fluorescent Probes Used for Conjugation to Biomolecules by Capillary Zone Electrophoresis

Abstract

The conjugation of three amine reactive fluorescent probes, each containing the fluorophore fluorescein but different reactive moieties, was compared using the protein myoglobin and the amino acid L-lysine as reagents. The three different reactive moieties were an isothiocyanate group (FITC), a succinimidyl ester group (CFSE), and a dichlorotriazine group (DTAF). The relative performance was based on the degree of conjugation to myoglobin, the rate of reaction, freedom from hydrolysis, and the stability of a conjugate with lysine. Performance was evaluated by separating the conjugation reaction reagents and products on-line, using capillary zone electrophoresis, and assessing relative amounts by absorbance detection. Each of the reactive probes demonstrated the ability to achieve a similar degree of conjugation, which depended mostly on allowed conjugated reaction time, and a rate of conjugation that rendered hydrolysis of the reactive moiety insignificant. For the relative rate of conjugation between probes and stability of the resulting conjugate, CFSE demonstrated superior performance, followed by DTAF and then FITC, for both the protein myoglobin and the amino acid L-lysine. The FITC conjugation reaction was much easier to control, however, which may be significant for applications that require a precise degree of conjugation. With regard to conjugate-bond stability, the FITC conjugate demonstrated inferior performance when subjected to incubation at 37 degrees C.

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with <em>Mycobacterium bovis</em> Bacille Calmette-Gu&#233;rin

“…The molecular basis of CFSE labeling is similar to FITC, which is also an amine-reactive fluorescent dye. CFSE is however preferred over FITC for conjugation as it creates very stable carboxamide bonds 16 . Moreover, CFSE is highly membrane permeable and passively diffuses into cells.…”

Vinyl Sulfone: A Multi-Purpose Function in Proteomics

“…Sulfonyl chlorides (2) are highly reactive but are also unstable in water (Lefevre et al, 1996), specially at the high pH required for the reaction with aliphatic amines, and they can also react with phenols (tyrosine), aliphatic alcohols (serine, threonine), thiols (cysteine) and imidazole (histidine). Isothiocyanates (3) are stable in water although their reactivity is only moderate and the degradation of the resulting thiourea has been reported (Banks & Paquette, 1995). In addition, the optimal pH needed for the reaction with lysine of these reagents (pH 9-9.5) is higher than for the formation of succinimidyl esters (pH 8-9) and may be unsuitable for modifying alkaline-sensitive proteins.…”

Preparation and Characterization of PEGylated Amylin

“…Both SDS-PAGE gels were run in parallel in the same electrophoresis system, and the top and bottom parts of the gel in these images are made visible for the sake of clarity. d, e Images from the gel stained with Coomassie Brilliant Blue for polypeptide moiety identification; f, g images from the same gel that were subsequently stained with barium/iodine for PEG moiety identification b display preferential reactivity with primary amine groups, although succinimidyl derivatives have also been shown to conjugate to hydroxyl groups such as those found in serine, tyrosine, and threonine (32)(33)(34)(35).…”