Fluorescein reagent

Once the protein is modified to contain an alkynyl group at its C-terminal it can be used to covalently link to its click chemistry reactant partner, an azide on the surface of an array. Other azido molecules also can be conjugated with an alkyne-protein to facilitate the detection or capture of the protein using affinity techniques. For instance, an azido-fluorescein reagent can be used to detect fluorescently the expressed protein in complex samples or an azido-biotin... 

The fluorescein reagent consists of a saturated solution of fluorescein in 50 per cent alcohol. 

Fluorescein reagent. To 1 g fluorescein, C20Hl2O5 (Colour Index 45350), add a mixture of 50 ml ethanol and 50 ml water. Shake, allow to stand for 24 hours, and filter the solution. 

Streptavidin-fluorescein reagent (Amersham, Little Chalfont, UK). 

The most used EIA reagents conjugate a fluotophote such as fluorescein-isothiocyanate (EITC) or thodarnine—isothiocyanate to antibody (or antigen) free amino groups. Examples of other commonly used fluotophotes for EIA and their spectral characteristics ate presented in Table 3. EIA assays ate available in sandwich and competitive formats similar to EIAs. Unlike EIA kits which can be used directly with visual color deterrnination, EIAs require a fluorometer, and thus ate primarily laboratory-based. 

The following are amongst the reagents that have been reported as being added to the mobile phase acids for quinine alkaloids [184], ninhydnn for amino acids [185 — 187], fluorescamine for biogenic amines [188] Fluorescein sodium [189], dichlorofluorescein [190], rhodamine 6G [191], ANS reagent [192] and bromine [193] have all been descnbed as additives to mobile phases... 

Note The full fluorescence intensity usually only develops about 30 min after the dipping process it then remains stable for several days if the chromatograms are stored in the dark (1, 5]. Fluorescein sodium can be employed in the reagent in place of 2, 7 -dichlorofluorescein [5]. The detection limits lie in the lower nanogram to picogram range [1, 5]. 

Muramoto K, Nokihara K, Ueda A, Kamiya H (1994) Gas-phase microsequencing of peptides and proteins with a fluorescent Edman-type reagent, fluorescein isothiocyanate. Biosci Biotechnol Biochem 58 300-304... 

The fluorescent properties of NHS-fluorescein are similar to FITC. The wavelength of maximal absorbance or excitation for the reagent is 491nm and its emission maximum is 518nm, exhibiting a visual color of green (Sheehan and Hrapchak, 1980). Its molar extinction coefficient at 491 nm in a pH 8.0 buffer environment is Other components in... 

Fluorescein-5-maleimide is slighdy soluble in aqueous solutions above pH 6 ( lmM concentration). It may be dissolved in DMF at higher concentrations and a small addition of this solution made to an aqueous reaction mixture to initiate labeling. Do not exceed 10 percent DMF in the reaction buffer to avoid protein precipitation. At pH 8, the reagent has an extinction coefficient at 490nm of about 78,000M 1 cm-1. 

Figure 9.9 SAMSA-fluorescein contains a protect thiol that can be deblocked by treatment with hydroxy-lamine. The reagent then can be used to modify molecules containing sulfhydryl-reactive groups.

SAMSA-fluorescein is an orange solid compound. Dissolved in buffer at pH 9.0, its maximal wavelength of absorption or excitation is at 495 nm, while its emission wavelength maximum is 520 nm. The reagent and all solutions and derivatives made from it are light sensitive and should be stored in the dark. SAMSA-fluorescein is soluble in aqueous solutions above pH 6.0, but it can be dissolved in DMF to prepare a concentrated stock solution prior to adding a small amount to a buffered reaction mixture. 

Fluorescein-5-thiosemicarbazide is soluble in DMF or in buffered aqueous solutions at pH values above 7.0. The reagent may be dissolved in DMF as a concentrated stock solution before adding a small aliquot to an aqueous reaction medium. The compound itself and all solutions made with it should be protected from light to avoid decomposition of its fluorescent properties. 

Figure 9.11 This carbohydrazide-containing fluorescein derivative can be used to modify aldehyde-containing molecules. Glycoconjugates may be labeled with this reagent after treatment with sodium periodate to produce aldehydes.

The methods for using this reagent in labeling glycoproteins on cell surfaces or in solution are similar to those described for fluorescein-5-thiosemicarbazide, above. 

Ahn, B., Rhee, S.G., and Stadtman, E.R. (1987) Use of fluorescein hydrazide and fluorescein thiosemicar-bazide reagents for the fluorometric determination of protein carbonyl groups and for the detection of oxidized proteins on polyacrylamide gels. Anal. Biocbem. 161, 245-257. 

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