| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| Other Sizes |
| Targets |
The primary target of N-aminofluorescein is not a biological receptor but rather aldehyde and ketone functional groups present on biomolecules. The amino group at the lactone nitrogen reacts specifically with carbonyl groups (aldehydes and ketones) under mild acidic conditions (pH 4-6) to form stable hydrazone linkages. This property allows for the fluorescent labeling of oxidized sugars, glycoproteins, aldehydic residues in DNA (e.g., apurinic/apyrimidinic sites), and other carbonyl-containing molecules. The compound is a chemical tool for bioconjugation and detection, not a therapeutic drug.
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| ln Vitro |
Advice (This is our suggested protocol, which should be adjusted to suit your particular circumstances as it simply offers guidance). N-aminofluorescein is made up of a hydrazide group and a fluorescein moiety that together can bind and identify Cu2+ to facilitate the hydrolysis of amide [1]. In a 70% HEPES buffer solution (pH 7.4) containing Cu2+, N-Aminofluorescein (FG) exhibits selectivity for Cu2+ and exhibits absorption and emission bands at 632 nm and 515 nm [2]. Advice (This is our suggested protocol, which should be adjusted to suit your particular circumstances as it simply offers guidance). Steps to determine Cu2+ in general [1]: 1. Make a 1.0 mM spiral fluorescein hydrazide ethanol stock solution; 2. Use 10 μM N-aminofluorescein and 0.01 M Tris-HCl buffer (pH 7.2) to perform fluorescence. Get the response going; 3. A suitable quantity of sample solution should be added to ensure that the final Cu2+ concentration does not surpass 10 μM. The final volume should be adjusted to 10 mL using 0.01 M Tris-HCl buffer (pH 7.2); 4. After two hours, transfer three milliliters of the solution to a one-centimeter quartz cell. Measure the fluorescence spectra and intensity at room temperature (λex/em = 495/516 nm), with excitation and emission slit widths of five nm each. Simultaneously prepare a blank solution devoid of Cu2+ and measure it under identical conditions for comparative analysis.
In vitro, N-aminofluorescein is used to label aldehyde- or ketone-containing biomolecules, such as oxidized glycans, glycosaminoglycans, or synthetic aldehydic probes. The labeling reaction takes place in aqueous buffers (0.1 M sodium acetate, pH 5.0) at room temperature for 2-6 hours. The resulting hydrazone conjugate exhibits bright green fluorescence (Ex 494 nm, Em 520 nm), enabling detection by fluorescence microscopy, flow cytometry, or plate-based fluorometry. It can also be used to detect apurinic/apyrimidinic sites in DNA, which contain reactive aldehyde groups, providing a tool for studying DNA damage and repair. |
| ln Vivo |
In vivo, N-aminofluorescein is not typically administered systemically as a therapeutic agent. However, it can be used to pre-label aldehyde- or ketone-functionalized biomolecules for later in vivo imaging studies. For example, nanoparticles or antibodies modified with ketone groups can be labeled with N-aminofluorescein ex vivo and then injected into animals to track their distribution. The green fluorescence allows for tissue localization using fluorescence imaging. Alternatively, it can be used as a probe for detecting oxidized products in fixed tissues, but its utility in live animal imaging is limited due to its relatively short wavelength (green) and potential for autofluorescence.
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| Enzyme Assay |
For non-cell-based labeling assays, a standard protocol is used for hydrazone formation. An aldehyde- or ketone-containing compound (e.g., pyridoxal phosphate, 0.1-1 mM) is dissolved in 0.1 M sodium acetate buffer (pH 5.0). N-aminofluorescein (dissolved in DMSO to 10 mM) is added to a final concentration of 0.5-2 mM. The reaction mixture is incubated at 37degC for 2-4 hours protected from light. The formation of the hydrazone product can be monitored by TLC (silica gel, ethyl acetate/methanol 9:1) or by reversed-phase HPLC with fluorescence detection (Ex 494 nm, Em 520 nm). The labeled product can be purified by preparative HPLC or by precipitation. For labeling of oxidized antibodies, the antibodies are first treated with sodium periodate to generate aldehyde groups, then reacted with N-aminofluorescein.
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| Cell Assay |
For in vitro cell-based assays, cells are first exposed to a stimulus that generates aldehyde-containing biomolecules, such as oxidative stress (H2O2 treatment) or glycosidase treatment to expose aldehyde groups on glycoproteins. Cells are fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and then incubated with 10-100 uM N-aminofluorescein in 0.1 M sodium acetate buffer (pH 5.0) for 1-2 hours at room temperature, protected from light. After washing with PBS, cells are mounted with DAPI-containing mounting medium and imaged by fluorescence microscopy (Ex 488 nm, Em 520-540 nm). The green fluorescence indicates the presence of aldehyde- or ketone-containing molecules. Alternatively, live cells can be labeled with the compound at pH 6.0-6.5 for 30-60 minutes, followed by washing and imaging.
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| Animal Protocol |
For in vivo animal studies, N-aminofluorescein is not widely used for direct animal injection. However, it can be used in ex vivo labeling studies. For example, a mouse model of oxidative stress (e.g., hepatic ischemia-reperfusion injury) is used. After the experiment, the animal is euthanized, and the target organ (e.g., liver) is perfused, fixed, sectioned, and then stained with N-aminofluorescein (10-100 uM in acetate buffer, pH 5.0) for 1-2 hours at room temperature. After washing, the sections are imaged using a fluorescence microscope with FITC filter set to detect aldehydic damage products. Control sections can be pre-treated with sodium borohydride to reduce aldehyde groups, confirming specificity. The compound can also be used to label aldehyde-modified liposomes or nanoparticles ex vivo prior to injection to track their biodistribution.
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| ADME/Pharmacokinetics |
N-Aminofluorescein has a molecular weight of 347.33 g/mol and a molecular formula of C20H13NO5. The compound is a pale orange to reddish powder. It is soluble in DMSO (10-25 mg/mL) and in ethanol but has low solubility in aqueous buffers. For in vitro labeling, a stock solution (10-20 mM) is prepared in DMSO, and then diluted into aqueous buffer (final DMSO concentration ≤5%). The compound exhibits excitation maximum at 494 nm and emission maximum at 520 nm (green), with an extinction coefficient of approximately 70,000 M-¹cm-¹. It is stable when stored at -20degC, protected from light and moisture. In solution, it is stable for several weeks at -20degC but may hydrolyze at neutral to alkaline pH. The hydrazone bonds formed are stable under physiological conditions but can be cleaved at very low pH (<2) or high pH (>10).
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| Toxicity/Toxicokinetics |
As a fluorescent labeling reagent, N-aminofluorescein is not intended for human use. No specific toxicology data is available. The compound is generally handled as a potential irritant; standard chemical safety precautions (gloves, lab coat, safety glasses) should be used. Avoid inhalation of dust. In cell culture, the compound is used at concentrations of 10-100 uM with no significant cytotoxicity reported. The fluorescein core is generally considered low in toxicity. However, the compound should be treated as a research chemical, and proper disposal methods should be followed.
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| References | |
| Additional Infomation |
N-Aminofluorescein is a research compound and is not approved for clinical use. It is a member of the fluorescein family of dyes and is specifically designed for the fluorescent labeling of carbonyl-containing molecules via hydrazone formation. This property makes it useful for detecting and quantifying aldehydes and ketones in complex biological samples, including oxidized proteins, advanced glycation end-products (AGEs), and damaged DNA. It is also used in the labeling of glycans after oxidative cleavage of their vicinal diols with periodate. The compound is an essential tool in chemical biology and glycobiology for studying oxidative stress, glycan structure, and cellular metabolism. It is for research use only.
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| Molecular Formula |
C20H14N2O4
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|---|---|
| Molecular Weight |
346.3362
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| Exact Mass |
346.095
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| CAS # |
98907-26-7
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| PubChem CID |
15883728
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| Appearance |
Off-white to light yellow solid powder
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| LogP |
3.463
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
0
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| Heavy Atom Count |
26
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| Complexity |
558
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1=CC=C2C(=C1)C(=O)N(C23C4=C(C=C(C=C4)O)OC5=C3C=CC(=C5)O)N
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| InChi Key |
CHYVTSCIBXXQJT-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C20H14N2O4/c21-22-19(25)13-3-1-2-4-14(13)20(22)15-7-5-11(23)9-17(15)26-18-10-12(24)6-8-16(18)20/h1-10,23-24H,21H2
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| Chemical Name |
2-amino-3',6'-dihydroxyspiro[isoindole-3,9'-xanthene]-1-one
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~125 mg/mL (~360.92 mM)
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.8873 mL | 14.4367 mL | 28.8734 mL | |
| 5 mM | 0.5775 mL | 2.8873 mL | 5.7747 mL | |
| 10 mM | 0.2887 mL | 1.4437 mL | 2.8873 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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