TRITC (Tetramethylrhodamine isothiocyanate) is a prominent member of the family of rhodamine dyes. Its high brightness and fluorescence quantum yield along with its capacity to covalently conjugate to proteins and polysaccharides efficiently under relatively mild conditions, render this rhodamine dye an indispensable fluorescent labeling agent.
Structure and physical properties
TRITC exhibits an excitation maximum at λ = 560 nm and emission maximum at approximately λ = 590 nm in DMSO solutions. The color of the compound is dark red while the emitted light is orange. There are two different isomers of TRITC:
Isomer I, also referred to as tetramethylrhodamine 5-isothiocyanate or 5-TRITC
Isomer II, also referred to as tetramethylrhodamine 6-isothiocyanate or 6-TRITC
The variation of the excitation and emission wavelengths among the two isomers of TRITC is minor. The absorption band observed in DMSO solutions exhibits a shoulder between 530-550 nm which is attributed to the coexistence of two isomers (Fig 1). TRITC-functionalized biopolymers such as TRITC- dextrans exhibit similar excitation and emission.
This fluorescent dye is currently available in the form of a mixture of two isomers (5- and 6- isothiocyanate isomers). Depending on the isomer, TRITC contains an isothiocyanate group at either position 5 (isomer I) or position 6 (isomer 2) of the bottom benzene ring, see figure 2. Isothiocyanate easily reacts with nucleophiles such as amines under mild conditions. The compound is often used in the form of one of its two isomers or sometimes as a mixture of the two.
Storage and stability
TRITC should be stored in a dry environment. When desiccated, it can be safely stored at room temperature. However, it is generally recommended to store it at temperatures between 4-8°C.
TRITC (isomer mix.) is delivered as a dark red powder, that is highly soluble in polar organic solvents such as DMSO and DMF. It also exhibits significant solubility in alcohols such as methanol and ethanol. However, it is incompatible with strong oxidizing agents.
TRITC can efficiently and covalently conjugate to -NH2 containing biomolecules such as proteins and antibodies or biorelevant macromolecules such as modified polysaccharides (e.g. lysine-dextran) at mild basic conditions. The rhodamine is thus conjugated to the macromolecules via a thiourea linker. TRITC can also label dextran via a well-developed TdB Labs methodology allowing for TRITC-dextrans with high degree of substitution.
TRITC and TRITC-labelled products are highly important for a range of biorelevant applications e.g. cell imaging, permeability and microcirculation studies, as well as research on drug delivery as a molecular size marker and in various biosensor applications.
Moreover, TRITC exhibits a strong orange fluorescence that is nearly insensitive to pH changes.
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