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The Thermo Scientific Pierce TRITC and NHS-Rhodamine Labeling reagents are ideal for labeling antibodies or other proteins and subsequent excess dye removal. NHS-Rhodamine is an amine-reactive fluorescent labeling reagent that absorbs green visible light (542 nm) and emits orange-red visible light (575 nm). Rhodamine displays lower sensitivity to pH than fluorescein and is more photostable; however, rhodamine conjugates have a significantly lower quantum yield than fluorescein conjugates.
N-Hydroxysuccinimide (NHS)-ester labeling reagents are the simplest and most commonly used reagents for labeling proteins. NHS esters react efficiently with primary amino groups (-NH2) in pH 7-9 buffers to form stable, covalent amide bonds. TRITC is the base tetramethylrhodamine molecule functionalized with an isothiocyanatereactivegroup(-N=C=S) at one of two hydrogen atoms on the bottom ring of the structure. Proteins, including antibodies, generally have several primary amines in the side chain of lysine (K) residues that are available as targets for NHS-ester and TRITC reagents.
The dye purification resin and spin columns included in the kit, eliminate equilibration steps and the need to collect and monitor gravity-flow fractions. This system enables efficient removal of excess fluor and, therefore, accurate determination of the dye-to-protein ratio and exceptional protein recovery.
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