Labeling with Lissamine rhodamine

Figure 23.7 Avidin (or (strept)avidin) can be labeled with Lissamine rhodamine sulfonyl chloride to form a fluorescent probe.

The following protocol is a general guide for labeling biological macromolecules with Lissamine rhodamine B sulfonyl chloride. Optimization of the fluorophore incorporation level (F/P ratio) may have to be done for specific labeling experiments. 

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. 

Lissamine rhodamine B sulfonyl hydrazine is a hydrazide derivative of sulforhodamine B that can spontaneously react with aldehyde- or ketone-containing molecules to form a covalent, hydrazone linkage (Fig. 217). It also can be used to label cytosine residues in DNA or RNA by use of the bisulfite activation procedure (Chapter 17, Section 2.1). The resulting fluorescent derivative exhibits an excitation maximum at a wavelength of 556 nm and a maximal emission wavelength of 580 nm when dissolved in methanol. In... 

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. 

Finally, another group of derivatives are the aldehyde-/ketone-reactive probes. This group is based on the activation of a sulfonyl hydrazine group of carbon number 5. They are based on Lissamine and Texas Red structures and used to label aldehyde-/ketone-containing molecules (with sugars). The most common aldehyde-/ ketone-reactive probes are Lissamine rhodamine B hydrazine and Texas Red hydrazine. 

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