Introduction: What is FAM dye & why it matters

6‑Carboxyfluorescein (6-FAM or FAM) is a fluorophore based on a fluorescein and a carboxyl group that can be used for functionalization with chemical moieties that allow for efficient and mild covalent attachments to biomolecules. The dye has an excitation maximum near 495 nm and emission around 520 nm and it delivers bright, stable fluorescence with high quantum yields and is compatible with detection channels usable for FITC, ATTO 488, Alexa Fluor 488, and DyLight 488.

FAM dyes are chemically more stable than other dyes and therefore have a wider field of potential applications. They can be added to Oligonucleotides already during solid phase synthesis and do not need to be introduced post synthetically. Another advantage of FAM dyes is that they are more cost efficient in comparison to other dyes since the core of the dye can be synthesized by a simple condensation reaction of two educts.

Key Characteristics of FAM Dye

Spectral features & optical performance

To determine the performance and area of use for a dye, there are several key factors to be considered:

– Excitation and emission maxima: These values represent the specific wavelengths where the dye absorbs (excitation maximum) and emits (emission maximum) photons most efficiently. The emission maximum wavelength always has a higher number, which means the emitted photons contain less energy than the absorbed photons. The difference is caused by energy loss which is called Stokes shift. A smaller stokes shift means that less energy is lost in the process between absorbance and emittance.

– Quantum yield: This value represents the quotient of emitted photons to absorbed photons. A value of 1 would mean that for each absorbed photon a photon is emitted. The smaller the quantum yield is the more energy is lost during the process by non-radiative processes as the induction of molecular vibrations.

– Extinction coefficient: This coefficient is a value that indicates how strongly a fluorophore absorbs light at a certain wavelength. The higher the value the more photons can be absorbed and the more sensitive the dye is for absorbance measurements. Please note, that a high extinction coefficient alone is not sufficient for a strong fluorescent dye since the emission is also influenced by the quantum yield.

All these values can be influenced by external factors as pH or the solvent which is used to dissolve the dye. Changes in pH can also lead to dramatic changes, as exemplary pH indicators change their color influenced by pH, or it even can lead to the destruction of the dye.

FAM is marked through its high quantum yield (>0.9), which overcompensates for a smaller extinction coefficient than other dyes of different wavelengths achieve. This is combined with high chemical stability over a wide pH range while the excitation and emission maxima stay almost constant. Please note that a lower pH does not lead to shift in these values, but the extinction coefficient strongly decreases for acidic pHs.

Reactive forms & conjugation options

The free 6-carboxy group of the FAM dye enables the introduction of several functional groups. This is done because a carboxyl group is not a good reactive group and labeling reaction utilizing a carboxyl-functionalized FAM would require harsh conditions and would provide low yields. Therefore, to produce a dye that could be used for labeling reaction in biochemistry, which require mild conditions and high yields, different functional groups are introduced. A common technique is to activate the carboxyl group by transforming it to e.g. a NHS ester, which can react with amines to give dye-labeled products. But since NHS esters require slightly basic pH, long reaction times and are sensitive to hydrolysis, alternatives were searched and found in introducing organic azides for labeling reactions. 6-FAM azide can be used for labeling reactions utilizing the Nobel prize winning Click Chemistry technology which can be performed in short times, at neutral pH at an ambient temperature. The azide can either be reacted in combination with alkynes under copper catalysis or without a catalyst in combination with strained alkynes which are available e.g. in DBCO or BCN groups.

Learn more about Click Chemistry here: Click Chemistry Glossary

Performance in imaging & detection methods

FAM dyes can be analyzed with most machines used for dye detection in standard machines in a biochemistry work environment such as fluorescence microscopy, flow cytometry and be used for probe detection.

–Fluorescence microscopy: FAM dye is usable for fluorescence microscopy as shown in our EdU cell proliferation kits for Imaging. All 488 variants of these kits use FAM based dyes for fluorescence labeling of de novo synthesized DNA. Due to its spectral properties, it can be used with a standard FITC filter set. For ideal experimental results for your own experiments a few things have to be considered. First, biological samples tend to produce autofluorescence in the green spectrum, where the FAM dye is active. Therefore, proper control measurements and maybe even spectral unmixing are necessary for your experiments. Additionally, since this dye is susceptible to photobleaching at long illumination times, minimized exposure times should be planned. If your experiment is not possible with these restrictions, please consider the usage of different dyes. baseclick offers you a variety of alternative dyes or you can contact us for  our recommendation for the fluorophore selection for your experiment (support@baseclick.eu).

–Flow cytometry (FC): FAM dyes are widely used for FC as in baseclick 488 variant of its EdU cell proliferation Kits for flow cytometry. Other potential applications include Antibody labeling, nucleic acid labeling and general labeling of all probes suited for FC analysis. FAM fluorophores provide strong signal intensity and can be analyzed with the FITC channel. FC has short exposure times and therefore photobleaching is not a factor for the choice of dye. For a reduced background by non-specific binding using a high-efficient reaction like click chemistry is recommended for labeling of your probes.

Learn more about Flow Cytometry here: Flow Cytometry Glossary

–Probes: FAM dyes can be used for the design of various probes such as in qPCR or for hybridization assays. It is especially suited for all kinds of oligonucleotide-based assays as TaqMan probes, since FAM dye can be added during solid phase synthesis and does not need to be incorporated post synthetical as many other dyes. Additionally, FAM forms a pair with TAMRA dyes for creating molecular beacons, where TAMRA is working as a quencher by fluorescence resonance energy transfer (FRET). TAMRA can also be replaced by a quencher like a black hole quencher.

Applications of FAM dye

Molecular probes & diagnostics

FAM dyes are widely used dyes in biochemistry because they are strongly fluorescent and are compatible with most detection equipment. Additionally, they are cheaper than other dyes. FAM is exemplary used for real-time qPCR analysis, for molecular beacons or for diagnostic and hybridization assays.

–qPCR: In qPCR FAM is commonly used as a reporter dye by incorporation of FAM dye labeled dNTPs in probe-based detection assays as TaqMan.

–Molecular Beacons: Molecular beacons are hairpin-shaped Oligo probes where FAM can act as a reporter at one end of the Oligo and while a different quencher is placed on the other side of the Oligo. The hairpin structure is produced by internal base pairing. While the probe remains in the hairpin structure, no emission signal of FAM is detected through FRET quenching. But if the probe is able to bind to a target, FAM is separated from the quencher and emitted fluorescence can be detected.

-Diagnostic assays: FAM labeled Oligo probes are used in many clinical diagnostic assays to detect various diseases such as infectious diseases (e.g. influenza), genetic mutations or for pharmacogenomics testing. All assays have in common that small FAM labeled Oligos are produced that bind to certain genetic material to prove the availability of the material. Therefore, all diagnostic assays are based on hybridization of probes to a specific nucleic acid counterpart.

Imaging in cell biology & biochemistry

FAM dyes can be used to label various biomolecules for live cell imaging. Possible targets for labeling are mostly proteins/peptides and nucleic acids. Detection of FAM labeled biomolecules is usually performed by microscopy-based techniques such as microscopy imaging. Confocal imaging results usually in the best experimental data to produce 3D images.

– Peptides/proteins: Amino acid-based biomolecules are usually labeled via amino reactive groups as NHS ester dyes. These dyes can react with every single primary amine available in the side chain of amino acids as in lysine occurring in the peptide/protein. Therefore, just unspecific and potential multiple labeling is achievable. A much more controlled labeling alternative is utilizing click chemistry. Here, modified amino acids carrying an azide or an alkyne are incorporated to the peptide and afterwards the modified target is labelled highly efficient with a FAM dye carrying the suitable opposite moiety for the click chemistry reaction.

-Nucleic acids: For nucleic acid labeling there are certain strategies. First, a modified nucleoside as EdU can be fed to cells and is then incorporated in de novo synthesized nucleic acids. The modified nucleotide can afterwards be labeled with a corresponding dye for detection of the successful incorporation. Another possibility is the incorporation of FAM dyes to synthetically produced nucleic acid strands. The incorporation can be achieved by FAM labeled amidites during synthesis or post synthetical by NHS ester chemistry or click chemistry.

Learn more about EdU Staining here: EdU Staining Glossary

Bio-conjugation & click-chemistry applications

FAM dyes are commonly used for the labeling of biomolecules. One of the most efficient, environmentally friendly methods for labeling is using the azide alkyne cycloaddition also known as click chemistry. For learning more about this reaction protocol, please click here. For performing click chemistry with a FAM dye, a specific reactive group for click chemistry is required as an azide. Azides allow for reaction with alkynes via copper catalysis by CuAAC reaction, or for the reaction with strained alkynes as available e.g. in DBCO via SPAAC reaction. baseclick is offering a standard azido modified variant which is well suited for almost all applications including SPAAC but also a variant which allows for faster reaction at lower catalyst concentration. The lower catalyst concentration improves the live-cell compatibility of the reaction and also a lower background can be achieved. Therefore, this variant is also used in baseclick´s sensitive cell proliferation detection kit for imaging which produces a better signal to noise ratio than our standard cell proliferation kit. These sensitive kits are also available for flow cytometry and for high-throughput screening.

Practical workflow example using FAM dye

If you have decided to perform a dye labeling reaction and have decided for FAM as the dye of choice, there are certain steps to determine which reaction should be used for labeling.

• Identify the target and check which functional groups are available for labeling or which functional groups can easily be introduced by modification. Check the stability of your target and analyze which reactions are possible for your target without the risk of decomposition.
• Choose a labeling reaction and check if a FAM dye with the corresponding reaction partner is available. Check for potential purification of the reaction product if needed.
• Select a method of analyzation which is suitable for your target and available detection system.
• Select a protocol with a similar reaction as a starting point of your labeling reaction.
• Perform your labeling reaction, do the purification if needed and analyze your results.

Key criteria when selecting a FAM dye for your experiments

There are several criteria to be considered before the choice for FAM to be used in your experiment should be made.

• Are the spectral properties of the dye (excitation at around ~ 495 nm, emission at around ~ 520 nm) suitable for your detection system (fluorometer, qPCR machine)?
• Do you have a filter set for a 488 dye available (microscopy imaging, flow cytometry)?

Afterwards, you need to check if the performance of FAM suits your experiment (extinction coefficient, quantum yield) and if you may need prolonged light exposure because of FAM being less photostable than other dyes. Please note that more photostable or brighter dyes a usually more expensive while being worse in the other category. Meaning a more photostable dye usually has less brightness and vice versa.

Then you should check the chemical stability of FAM for the conditions you consider. The dye is relatively stable and suited for most labeling reactions, but the fluorescence is strongly reduced in acidic conditions. If multiplexing experiments are planned, check that FAM or other fluorophores used to not interfere which each other’s excitation and emission area. At least you should check if autofluorescence at green light needed for excitation of FAM can be an issue for your experiment. After considering all these parameters, if FAM is possible for your experiment, you should always consider a FAM dye because of its wide availability at cheap prices.