| Size | Price | Stock | Qty |
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| 100mg |
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| 250mg |
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| 500mg |
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| Targets |
5-FAM itself does not have a specific biological target. As a fluorescent dye, its "targets" are specific functional groups (such as primary amines -NH₂ or thiols -SH) present in molecules, to which it forms stable covalent bonds (e.g., amide or disulfide bonds) for labeling purposes. It can label amine-containing biomolecules following carbodiimide activation (e.g., with EDC/NHS) of its carboxyl group.
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| ln Vitro |
5-FAM includes a carboxylic acid that is activated by carbodiimide when it comes into contact with primary amines. The most popular fluorescent derivatization reagent for labeling biomolecules is fluorescein. Apart from its exceptional fluorescence quantum yield, superior water solubility, and relatively high absorption rate, fluorescein also exhibits an excitation maximum that is quite similar to the argon ion laser's 488 nm spectral line [1].
In vitro activity assessment focuses on its effectiveness as a labeling reagent, rather than biological activity. Molecules labeled with 5-FAM can be visualized and tracked using techniques like fluorescence microscopy and flow cytometry. One study used a 5-FAM-labeled peptide substrate in a high-throughput MMP-9 anticancer assay. Additionally, 5-Carboxyfluorescein diacetate (5-CFDA) can be hydrolyzed by intracellular esterases to yield fluorescent 5-FAM, a process used to assess cell viability. Its advantages include excellent fluorescence quantum yield, good water solubility, and an excitation maximum that closely matches the 488 nm line of argon-ion lasers. |
| ln Vivo |
5-FAM is generally not recommended for in vivo imaging. For most in vivo applications, near-infrared dyes are preferred due to superior tissue penetration. In vivo studies have primarily utilized 5-FAM as a tracer to study its sustained release properties in formulations like vesicular phospholipid gels (VPGs) for drug delivery systems. For example, one study showed that 5-FAM-labeled thymopentin (5-FAM-TP5) encapsulated in VPGs had a release time of up to 216 hours after subcutaneous injection.
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| Enzyme Assay |
The peptide substrate containing 5-FAM (donor) and QXL520 (acceptor) was used. Upon cleavage of the peptide by MMP-9, the fluorescence of 5-FAM was recovered and detected at excitation/emission = 485 nm / 525 nm using a multi-plate reader. [1]
For inkjet printing on parchment paper, the fluorescence of 5-FAM was measured at excitation/emission = 485 nm / 525 nm using a fluorescence microscope. Images were analyzed using MetaMorph software. [1] |
| Cell Assay |
Used to track the cellular uptake and distribution of labeled molecules. A basic protocol is as follows:
1. Cell Preparation: Seed cells into suitable dishes (e.g., confocal dishes) and culture to the desired density. 2. Labeled Molecule Treatment: Treat cells with the target molecule (e.g., siRNA, peptide, or antibody) pre-labeled with 5-FAM. Fluorescent signals can often be observed within 4 hours. 3. Incubation: Incubate in a cell culture incubator for 1-24 hours, depending on the uptake rate of the target molecule. 4. Wash and Fix (Optional): Wash cells with PBS to remove unbound or free label. Fix with 4% paraformaldehyde if imaging. 5. Detection: Visualize using a fluorescence microscope (FITC channel), flow cytometer, or fluorescence plate reader. Use excitation at ~490-495 nm and detect green emission at ~520 nm. |
| Animal Protocol |
Primarily used to study the pharmacokinetics of drug delivery systems rather than assessing direct in vivo activity. Using subcutaneous injection as an example:
1. Animal Preparation: Select an appropriate strain of mice. 2. Administration: Administer the formulation containing the 5-FAM-labeled drug (e.g., 5-FAM-TP5 solution or VPGs gel) via subcutaneous injection. 3. Sample Collection: Collect blood samples or dissect tissues at predetermined time points (e.g., from 0.5 hours to several days post-injection). 4. Sample Processing: Process blood samples to obtain plasma and extract the 5-FAM-labeled compound using methods like protein precipitation. 5. Detection and Analysis: Quantify the concentration of the labeled compound in samples using a fluorescence spectrophotometer or LC-MS/MS. Generate a concentration-time curve and calculate pharmacokinetic parameters. |
| ADME/Pharmacokinetics |
The pharmacokinetics of 5-FAM itself are often used as a reference for evaluating its drug delivery systems. Research indicates that 5-FAM is typically cleared rapidly. In a study comparing free 5-FAM-TP5 solution with a vesicular phospholipid gel (VPGs) formulation in mice, free 5-FAM-TP5 solution showed a fast PK profile, reaching a Tmax of 0.5 hours and a short half-life (t₁/₂) of 2.69 hours. In contrast, the VPGs formulation achieved significant sustained release, prolonging the half-life to 114 hours and increasing the mean residence time (MRT) to over 190 hours.
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| Toxicity/Toxicokinetics |
According to Safety Data Sheets provided by suppliers, 5-FAM is classified as harmful (Xn). However, at commonly used concentrations (e.g., 1-10 µM for cell labeling), literature reports it exhibits low cytotoxicity. While it has irritating potential and standard laboratory safety precautions (e.g., wearing gloves, lab coat, and goggles) are necessary, it is not an acutely toxic compound.
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| References | |
| Additional Infomation |
5-Carboxyfluorescein is a monocarboxylic acid that functions as a fluorescent dye, and its structure is related to that of fluorescein (lactone form).
5-FAM (5-carboxyfluorescein) serves as the fluorescence donor in the FRET peptide substrate. The peptide sequence is Pro-Leu-Gly-Cys-His-Ala-Arg-Lys, with 5-FAM attached at the lysine residue and QXL520 (acceptor) at the N-terminus. The peptide is specifically cleaved by MMP-9 at the Gly-Cys bond, leading to separation of the donor and acceptor and increase of 5-FAM fluorescence. [1] |
| Molecular Formula |
C21H12O7
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| Molecular Weight |
376.32
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| Exact Mass |
376.058
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| CAS # |
76823-03-5
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| Related CAS # |
5-FAM SE;92557-80-7
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| PubChem CID |
123755
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.7±0.1 g/cm3
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| Boiling Point |
725.2±60.0 °C at 760 mmHg
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| Melting Point |
368-372°C
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| Flash Point |
266.9±26.4 °C
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| Vapour Pressure |
0.0±2.5 mmHg at 25°C
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| Index of Refraction |
1.816
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| LogP |
2.62
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
1
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| Heavy Atom Count |
28
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| Complexity |
637
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
NJYVEMPWNAYQQN-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C21H12O7/c22-11-2-5-15-17(8-11)27-18-9-12(23)3-6-16(18)21(15)14-4-1-10(19(24)25)7-13(14)20(26)28-21/h1-9,22-23H,(H,24,25)
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| Chemical Name |
5-Carboxyfluorescein 3',6'-Dihydroxy-3-oxo-spiro[isobenzofuran-1(3H),9'-[9H]xanthene]-5-carboxylic acid
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| Synonyms |
5-FAM 5-Carboxyfluorescein 5 Carboxyfluorescein 5Carboxyfluorescein5 FAM 5FAM
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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 : ≥ 31 mg/mL (~82.38 mM)
H2O : ~1 mg/mL (~2.66 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.64 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (6.64 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.6573 mL | 13.2866 mL | 26.5731 mL | |
| 5 mM | 0.5315 mL | 2.6573 mL | 5.3146 mL | |
| 10 mM | 0.2657 mL | 1.3287 mL | 2.6573 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.
| NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT03850132 | COMPLETED | Behavioral: FAM-SOTC-PL | Bereavement | University of Iceland | 2014-07-27 | Not Applicable |
| NCT02815462 | WITHDRAWN | Other: FAMFACESG Other: Control Other: FAMFACESG |
Patient Readmission | Singapore General Hospital | 2016-08 | Not Applicable |
| NCT03901027 | COMPLETED | Behavioral: Therapeutic Conversation Interventions | Chronic Illnesses | University of Iceland | 2015-03-01 | Not Applicable |
| NCT06114186 | RECRUITING | Device: FAM intervention consisting of a Follower of glucose data, Action Plan, and Remote Monitoring of glucose data Other: standard of care |
Type 1 Diabetes | University of Minnesota | 2024-01-16 | Early Phase 1 |
| NCT04339465 | UNKNOWN STATUS | Behavioral: CARE-FAM Behavioral: WEP-CARE |
Rare Diseases | Silke Wiegand-Grefe, Prof. Dr. | 2019-01-01 | Not Applicable |
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