Lucifer Yellow fluorophores

Figure 9.41 The basic structure of Lucifer Yellow fluorophores.

The amine-reactive 5-(dimethylamino)naphthalene-l-sulfonyl (dansyl) chloride 28 [80] and related fluorophores [81, 82], as well as the 5-((2 aminoethyl)amino) naphthalene-1-sulfonic acid (EDANS) 29, are included in the naphthalene fluorophore family. Derivatives of the latter, such as compound 30, exhibit a Lm.ix/ Lem 336/520 nm, molar absorptivity (e) of 6.1 x 103 M-1 cm-1, and a fluorescent quantum yield of 0.27 in water [83], The use of EDANS is particularly interesting in FRET experiments [84, 85]. Furthermore, 4-amino-3,6-disulfonylnaphthalimides (e.g., Lucifer yellow 31), associated to a longer absorption (Lmax 428 nm) [86] are suitable polar tracers [87]. 

This fluorophore has excitation maxima at 375 and 400 nm and an emission maximum at 410 nm. The small Stoke s shift may create some difficulty in discrete excitation without contaminating the emission measurement with scattered or overlapping light. The extinction coefficient of the molecule in water is about 27,000M 1cm 1. Cascade Blue and Lucifer Yellow derivatives can be simultaneously excited by light of less than 400 nm, resulting in two-color detection at 410 and 530 nm. 

These fluorophores have excitation maxima at 377-378 nm and at 398-399nm and emission maxima at 422-423 nm. The extinction coefficients of the molecules in water are about 27,000 M 1cm 1. The Cascade Blue derivatives can be used along with Lucifer Yellow... 

One Lucifer Yellow derivative is available for labeling sulfhydryl-containing molecules. Lucifer Yellow iodoacetamide is a 4-ethyliodoacetamide derivative of the basic disulfonate aminonaph-thalimide fluorophore structure (Invitrogen). The iodoacetyl groups react with —SH groups in proteins and other molecules to form stable thioether linkages (Figure 9.42). 

The spectral characteristics of Lucifer Yellow iodoacetamide produce luminescence at somewhat higher wavelengths than the green luminescence of fluorescein, thus the yellow designation in its name. The excitation maximum for the probe occurs at 426 nm and its emission at 530 nm. The rather large Stoke s shift makes sensitive measurements of emission intensity possible without interference by scattered excitation light. The 2-mercaptoethanol derivative of the fluorophore has an extinction coefficient at pH 7 of about 13,000 M cm-1 at 426nm. 

Lucifer Yellow CH is soluble in aqueous solution, and it should be stable for awhile if protected from light. The reagent is available as three different salts of the sulfonate groups. The ammonium salt of the fluorophore is soluble to a level of 9 percent in water, while the lithium and potassium salts have a solubility of 5 and 1 percent, respectively. A concentrated stock solution of the fluorophore may be prepared in water and an aliquot added to a buffered reaction medium to facilitate the transfer of small quantities. For aqueous reactions, a pH range of 5-9 will result in efficient hydrazone formation with aldehyde or ketone residues. 

Lucifer Yellow derivatives are used extensively for cytochemical staining applications, especially in neurophysiology (Stewart, 1981a, b). The fluorophores are 3.6-disul-... 

The decay time of the fluorophor used as label should be longer than the rotational time of the hapten but shorter than the rotational time of the complex formed. Fluorescein, which has a fluorescence lifetime of 4.5 ns, or rhodamine are the most frequently used fluorophors. For larger antigens, fluorophors with somewhat longer decay times such as Lucifer yellow, dansyl, and umbelliferone derivatives have been tested. In a typical competitive format, the degree of polarization is inversely proportional to the concentration of the analyte. 

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