Fluorescein Lissamine derivatives

The spectral characteristics of protein conjugates made with Lissamine rhodamine B derivatives are of longer wavelength than those of tetramethylrhodamine—more toward the red region of the spectrum. In addition, modified proteins have better chemical stability and are somewhat easier to purify than those made from TRITC (discussed previously). Lissamine derivatives also make more photostable probes than the fluorescein derivatives (Section 1, this chapter). 

The intense Texas Red fluorophore has a QY that is inherently higher than the tetrameth-ylrhodamine or Lissamine rhodamine B derivatives. Texas Red s luminescence is shifted maximally into the red region of the spectrum, and its emission peak only minimally overlaps with that of fluorescein. This makes Texas Red derivatives among the best choices of labels for use in double-staining techniques. 

The red-emitting rhodamine derivatives are constructed around the same basic xanthene framework as is fluorescein (2). Tetramethylrhodamine isothiocyanate (TRITC) has been widely employed for immunofluorescence. Additional derivatives of rhodamine available for conjugation to antibodies include lissamine rhodamine sulfonyl chloride (RB-200-SC), rhodamine B isothiocyanate (RBITC), rhodamine X isothiocyanate (XRITC), and Texas... 

The red-emitting rhodamine derivatives are constructed around the same basic xanthene framework as is fluorescein (2). Tetramethyl-rhodamine isothiocyanate (TRITC) has been widely employed for immunofluorescence. Additional derivatives of rhodamine available for conjugation to antibodies include lissamine rhodamine sulfonyl chloride (RB-200-SC), rhodamine B isothiocyanate (RBITC), rhodamine X isothiocyanate (XRITC), and Texas Red (Molecular Probes, Inc.). The spectra of XRITC and Texas Red are shifted to longer wavelengths compared to those of other rhodamines, which makes them particularly useful for combination with fluorescein in dual-labeling procedures see Section 5, below). Of the two, Texas Red, which is more hydrophilic and less likely to precipitate proteins upon conjugation (12), is more commonly employed. 

To date, the most popular fluorescent labels for FIA have been those derived from the long-wavelength, strongly emitting xanthene dyes fluorescein isothiocyanate (FTTC) and lissamine rhodamine B (RB200).F The isothiocyanates or isocyanates of these fluorophores can be used to label primary and secondary aliphatic amines in aqueous solutions by simple procedures. 

Similar analogs to those described above were prepared by Lai and coworkers 382). These investigators used a 7-(aminocaproyl) linker to couple lissamine rhodamine B (structure E above) or fluorescein isothiocyanate to taxol, and confirmed that both derivatives can be used to carry out intracellular fluorescence mapping. 

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