Fluorescein is a common green-fluorescent derivatization reagent, while succinimidyl esters are frequently used for the labeling of the primary amines (R-NH2) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. This 'SFX' succinimidyl ester contains a seven-atom amino-hexanoyl spacer between the fluorophore and the reactive group. This spacer separates the fluorophore from the biomolecule to which it is conjugated, potentially reducing the quenching that typically occurs upon conjugation. The resulting fluorescein conjugates can be detected with standard FITC or GFP filter sets. Fluorescein-based dyes and their conjugates have a relatively high rate of photobleaching and a pH-sensitive fluorescence. Alexa Fluor® 488 and Oregon Green® 488 are alternative dyes whose spectra mimic those of fluorescein and are much more photostable with little to no pH sensitivity in the physicological pH range.
• Fluorophore label : fluorescein
• Reactive group: succinimidyl ester
• Reactivity: primary amines on proteins and ligands, amine-modified oligonucleotides
• Molecular weight: 586
• Extinction coefficient: 74,000 cm-1M-1
• Ex/Em of the conjugate: 494/520 nm
• Spectrally similar dyes: Alexa Fluor® 488, GFP
Typical Conjugation Reaction
Amine-reactive reagents can be conjugated with virtually any protein or peptide; the provided protocol is optimized for IgG antibodies. You may scale the reaction for any amount of protein, but the concentration of the protein should be at least 2 mg/mL for optimal results. We recommend trying three different degrees of labeling, using three different molar ratios of the reactive reagent to protein.
The fluorescein succinimidyl ester is typically dissolved in high-quality, anhydrous dimethylformamide (DMF) or dimethylsulfoxide (DMSO) and the reaction carried out in 0.1–0.2 M sodium bicarbonate buffer, pH 8.3, at room temperature for 1 hour. Because the pKa of the terminal amine is lower than that of the lysine epsilon-amino group, you may achieve more selective labeling of the amine terminus using a buffer closer to neutral pH.
Labeled antibodies are typically separated from free dye using a gel filtration column, such as Sephadex™ G-25, BioGel® P-30, or equivalent. For much larger or smaller proteins, select a gel filtration media with an appropriate molecular weight cut-off or purify by dialysis.