5-Aminofluorescein

5-Aminofluorescein (also called 5‑AF or Fluoresceinamine) is widely used as a fluorescent labeling reagent. The primary amine of 5-Aminofluorescein can react with carboxyl groups (via EDC/NHS activation), aldehydes, ketones, or activated esters (e.g., NHS esters) to form stable amide or imine linkages. It exhibits an excitation maximum at ~490 nm (λmax = 496 nm in borate buffer pH 8.5) and an emission maximum at ~515 nm, delivering a bright green signal with good photostability and a Stokes shift of about 40 nm. Its fluorescence intensity is pH‑dependent, increasing notably under alkaline conditions (pH 8‑9), which is attributed to changes in the protonation state of the amino group.

5-Aminofluorescein has found broad use in biological and medical research – from labeling proteins and antibodies to constructing targeted drug delivery systems and fluorescent sensors. 5-Aminofluorescein is also a key intermediate in the synthesis of fluorescein isothiocyanate (FITC Isomer I), one of the most popular reagents for protein labeling.

Product Parameters

Preparation & Storage Notes

5‑AF is typically synthesized starting from 4‑nitrophthalic acid, followed by reduction (e.g., with hydrazine/FeOOH) and condensation with resorcinol in methanesulfonic acid. The crude product is a mixture of 5‑ and 6‑isomers, which are separated by crystallization. Modern methods (microwave‑assisted or enzymatic) can improve yield and reduce by‑products. Final purification by HPLC ensures ≥95% purity, with structure confirmed by NMR and MS.

Storage – The compound is sensitive to light, air, and moisture. Store in a sealed, argon‑purged container at 4°C in the dark. For long‑term storage (>6 months), -20°C as a powder is recommended (stable for at least 3 years). Solutions in DMSO can be stored at -80°C for 6 months or at -20°C for 1 month. Avoid repeated freeze‑thaw cycles. Transport at ambient temperature is acceptable if protected from light and moisture.

Frequently Asked Questions (FAQ)

Q1: What is the difference between 5‑Aminofluorescein (5‑AF) and FITC? How do I choose?

A: Both are green‑fluorescent dyes, but their reactive groups differ.

● 5‑AF has a primary amine (–NH₂). It requires activation of a target’s carboxyl group (using EDC/NHS) before conjugation. Best for labeling molecules that already contain a carboxyl group.

● FITC has an isothiocyanate group (–N=C=S) that reacts directly with amines on the target (e.g., lysine residues in proteins) – no prior activation needed. FITC is more convenient for routine protein labeling.

● Choose 5‑AF if you need a specific, oriented conjugation (e.g., via a carboxyl handle) or if you plan to synthesize custom fluorescent derivatives. FITC is generally simpler for direct antibody labeling.

Q2: What are typical reaction conditions for labeling biomolecules with 5‑AF?

A: The most common method is amide coupling: activate carboxyl groups on the target molecule with EDC and NHS (in MES or PBS buffer, pH 5.5–6.5), then add 5‑AF and adjust to pH 7.5–8.5. Incubate at room temperature or 37 °C for 2–4 hours. For aldehyde‑containing targets (e.g., glycoproteins), use reductive amination with sodium cyanoborohydride. Unreacted dye can be removed by desalting or dialysis. A dye‑to‑protein molar ratio of 5:1 to 20:1 is a good starting point.

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Whether you need a small sample for a pilot experiment or bulk quantities for commercial production, Cosperpharm is ready to support you. Contact us today to discuss your requirements for 5-Aminofluorescein – and let’s see how we can help move your project forward.