Introduction to the AF Series Fluorescent Dyes: Overview, Applications, and Selection Guide
Introduction to the AF Series Fluorescent Dyes: Overview, Applications, and Selection Guide
Overview of AF Dyes
The AF series represents a new generation of fluorescent dyes derived from traditional fluorophore scaffolds such as rhodamines, cyanines, and coumarins, which have been extensively modified through high degrees of sulfonation.
By introducing multiple sulfonate groups (–SO₃⁻), the dye molecules become negatively charged and highly hydrophilic. This markedly improves their aqueous solubility compared with traditional dyes and reduces aggregation or precipitation after protein labeling.
The AF series covers a broad spectral range from UV to near-infrared, including AF 350, 405, 430, 488, 532, 546, 555, 568, 594, 647, 660, 680, 700, and 750, meeting the multicolor detection needs of most flow cytometry, fluorescence microscopy, and imaging platforms.
In the naming scheme “AF + number,” the number approximately corresponds to the dye’s main excitation/absorption maximum or a common laser line wavelength. For example:
- AF 488 is compatible with a 488 nm laser.
- AF 555 is compatible with 532/561 nm lasers.
- AF 647 is compatible with 633/640 nm lasers.
Spectrally, AF dyes closely match many commonly used traditional fluorophores, such as:
- AF 350 ≈ AMCA / AMCA-X
- AF 405 ≈ Cascade Blue / Pacific Blue
- AF 430 ≈ Lucifer Yellow, etc.
- AF 488 ≈ FITC, Cy2
- AF 555 / AF 568 ≈ Cy3, rhodamine-type dyes
- AF 594 ≈ Texas Red
- AF 647 / AF 660 ≈ Cy5
- AF 680 / 700 / 750 ≈ Cy5.5 / Cy7, etc.
Therefore, on instruments with existing filter sets and laser configurations, AF dyes can often directly replace traditional fluorophores without the need to modify the optical system.
Core Advantages of AF Dyes
1. High Brightness
AF dyes generally possess high molar extinction coefficients and moderate to high quantum yields. Many wavelengths show overall brightness significantly superior to traditional dyes:
- For example, AF 488 has a quantum yield of about 0.9. AF 568 and AF 594 also combine high quantum yields with large extinction coefficients.
Compared with traditional dyes such as FITC, Cy3, and Cy5, AF conjugates provide stronger signals at the same degree of labeling, which is advantageous for detecting low-abundance targets or shortening exposure times.
2. Excellent Photostability
Under continuous excitation, many AF dyes exhibit lower photobleaching rates, significantly extending usable imaging time in fluorescence microscopy and live-cell imaging.
- AF 555 has been shown to be more resistant to photobleaching than tetramethylrhodamine and Cy3.
- AF 647 and AF 568 are also widely used in demanding super-resolution techniques such as STED and STORM.
3. Broad pH Tolerance
For many AF dyes, fluorescence intensity remains essentially unchanged across pH 4–10 (e.g., AF 488, AF 405), which is a clear advantage over pH-sensitive dyes such as fluorescein.
This enables AF dyes to be used in acidic organelles, extracellular environments, and under certain fixation/mounting conditions while still maintaining stable signal.
4. Excellent Aqueous Solubility and Low Non-Specific Binding
The high polarity introduced by multiple sulfonate groups provides AF dyes and their conjugates with good solubility in aqueous media, reducing the need for organic cosolvents and minimizing problems caused by dye hydrophobicity, such as:
- Protein aggregation or precipitation.
- Dye–dye stacking and self-quenching.
- Non-specific hydrophobic adsorption.
This is particularly beneficial for antibody and protein conjugation, helping to maintain protein conformation and biological activity.
5. Instrument Compatibility and Multicolor Panel Design
The AF series was designed with compatibility to common laser lines (355, 405, 488, 532, 561, 594, 633/640 nm, etc.) in mind. Most dyes can be used directly on existing flow cytometers and fluorescence microscopes.
At the same time, dense wavelength coverage from the blue region to the near-infrared, combined with narrow-band filters, allows construction of multicolor panels with 8, 10, or even more colors.
Labeling Principles of AF Dyes
1. Random Lysine Labeling (NHS Ester)
The most common AF labeling strategy uses NHS esters (N-hydroxysuccinimide esters):
- Conditions: Typically carried out in a buffer at pH 7.5–8.5.
- Mechanism: The NHS ester is attacked by nucleophilic primary amines on proteins (ε-amine of lysine side chains or N-terminal amines), releasing N-hydroxysuccinimide and forming a stable amide bond, thereby covalently attaching the dye to the protein.
This method is simple and broadly applicable, suitable for routine preparation of most antibodies and proteins. However, because proteins differ in lysine number and surface distribution, the same process may lead to different degrees of labeling and varying impact on activity across different proteins, or even between batches of the same protein.
2. Thiol-Specific Labeling (Maleimide)
For proteins containing cysteine residues, maleimide-functionalized AF dyes can be used to react with free thiols via a Michael addition under mild conditions, forming a stable thioether bond:
- Suitable for reduced proteins or those engineered to introduce specific cysteine sites, enabling higher site specificity.
- Commonly used to generate highly homogeneous fluorescent antibodies and recombinant protein probes.
3. Click Chemistry Systems (Azide / Alkyne / DBCO / Tetrazine)
For projects requiring more precise site definition or a controlled linker architecture, bioorthogonal click reactions can be employed:
- Azide / Alkyne:
- Classical Cu(I)-catalyzed Huisgen cycloaddition (CuAAC).
- Suitable for in vitro labeling under non-physiological conditions.
2. DBCO / BCN and other strained alkynes:
- Copper-free click reactions (SPAAC) used for live-cell or in vivo labeling to avoid copper toxicity.
3. Tetrazine / TCO:
- Extremely fast inverse electron-demand Diels–Alder (IEDDA) reactions, ideal for real-time in vivo imaging and highly efficient conjugation.
By first introducing a corresponding “handle” (azide, alkyne, TCO, etc.) into the target protein, antibody, or cell surface, and then reacting with an AF dye bearing the complementary functional group, truly site-specific labeling can be achieved.
4. Importance of Site-Specific Labeling
In drug development for modalities such as CAR-T, ADCs, and bispecific antibodies, fluorescently labeled proteins are not merely required to “light up,” but also to:
- Maintain the affinity and specificity of the protein/antibody for its target.
- Achieve high batch-to-batch consistency to support method development and quality control.
- Place labeling sites away from functional domains to minimize structural interference.
Therefore, beyond conventional random NHS labeling, combining engineered amino acid sites with thiol- or click-based site-specific strategies is one of the key steps in obtaining high-quality AF-labeled proteins.
Typical Application Scenarios
Thanks to their high brightness, excellent stability, and multicolor capabilities, AF dyes have become mainstream fluorescent tools in life science research and biopharmaceutical development. Typical applications include:
1. Flow Cytometry
- Labeling of surface and intracellular proteins.
- Detection of CAR-T cell positivity (e.g., AF488/AF647-labeled antibodies).
- Immunophenotyping and multicolor panel design.
2. Fluorescence and Confocal Microscopy
- Immunofluorescence (IF) and labeling of subcellular structures such as cytoskeleton and mitochondria.
- IHC/IF on tissue sections, multiplexed staining using AF594/AF647, etc.
3. Super-Resolution Microscopy (STED / STORM / SIM)
- Far-red dyes such as AF647 and AF568 are suitable for STORM/STED.
- Short-wavelength dyes such as AF405 can be used for photoactivation/photo-conversion.
4. PK / Biodistribution and In Vivo Imaging
- Using near-infrared dyes such as AF680/700/750 to label antibodies, protein therapeutics, or nanocarriers for in vivo pharmacokinetics and tissue distribution studies in animals.
5. Cell Therapy and Biologics Development
- AF-labeled target proteins can be used as standards or detection reagents, playing key roles in CAR-T cell construction, potency evaluation, IHC distribution, and affinity assessment of bispecifics/ADCs.
List of Reactive AF Dye Derivatives
Series | Reactive Group Type | English Full Name | Aladdin Cat. No. | Grade & Purity | Key Features (Brief) | Typical Applications |
AF405 | NHS ester (amine-reactive) | AF 405 NHS ester | Ex: 401 nm Em: 422 nm | Blue AF405 activated ester; Ex/Em ≈ 401/422 nm; essentially stable at pH 4–10; good water solubility | Short-wavelength channel multicolor imaging; amine labeling of proteins/antibodies; flow cytometry with 405 nm violet laser | |
AF430 | Carboxylic acid | AF 430 carboxylic acid / AF 430 Acid | – | AF430 core + carboxylic acid; blue excitation, green-yellow emission; good water solubility | In-house preparation of activated esters; solid-phase coupling; multicolor imaging | |
AF430 | NHS ester (amine-reactive) | AF 430 NHS ester | – | Classical amine-reactive type; suitable to formulate “universal labeling reagents” in one step | Random lysine labeling of antibodies/proteins; cell staining; flow cytometry | |
AF430 | Amine | AF 430 amine | – | Terminal primary amine that can be further coupled to activated esters or aldehydes | Secondary derivatization; crosslinker assembly; surface modification of nanomaterials | |
AF430 | Maleimide (thiol-reactive) | AF 430 maleimide | – | Thiol-specific; AF430 used as donor/reporter dye | Site-specific Cys labeling; enzyme activity probes | |
AF430 | Azide (Click) | AF 430 azide | – | Azide functionality; suitable for CuAAC/SPAAC | Metabolic labeling and click staining; nucleic acid and carbohydrate probes | |
AF430 | Alkyne (Click) | AF 430 alkyne | – | Terminal alkyne; click linkage with azide substrates | Small-molecule tracing; click modification of polymers | |
AF430 | Tetrazine (Click) | AF 430 tetrazine | – | IEDDA click dye; fast reaction rate; no copper catalyst required | Copper-free click in cells/in vivo; live tracking | |
AF488 | Carboxylic acid | AF 488 carboxylic acid | – | – | Carboxylic acid form of the classic 488 nm green dye; good water solubility | Preparing in-house activated esters; coupling to small molecules; nanoparticle labeling |
AF488 | NHS ester (amine-reactive) | AF 488 NHS ester | – | Spectrum similar to FITC but brighter and more stable; first-choice amine-reactive dye | Antibody/protein labeling, flow cytometry, immunofluorescence | |
AF488 | NHS ester (amine-reactive, TEA salt) | AF 488 NHS ester (TEA salt) | – | TEA salt form of the classic AF488 NHS ester; bright green, photostable; forms stable amide bonds with primary amines at pH 7.5–8.5 | Labeling primary amines on antibodies/proteins/oligonucleotides; used in flow cytometry, immunofluorescence, confocal imaging | |
AF488 | NHS ester (amine-reactive, di-TEA salt) | AF 488 NHS ester (di-TEA salt) | – | Di-triethylamine salt of AF488 NHS ester, improving water solubility and formulation compatibility; reactivity equivalent to standard AF488 NHS ester | Same as above; used where higher water solubility or special buffer systems are required for amine labeling | |
AF488 | Primary amine (for further coupling) | AF 488 amine | – | AF488 dye with a terminal primary amine; can be further coupled to NHS esters, activated carboxylic acids, or aldehydes, enabling customized linkers and conjugates | In-house synthesis of conjugates; coupling to small molecules/peptides/polymers; surface modification of nanomaterials | |
AF488 | Click – Azide (azide, bis-TEA) | AF 488 azide, bis(triethylammonium) salt | ≥90% (HPLC) | Bis-triethylammonium salt; improved water solubility and batch-to-batch consistency | High-demand click labeling requiring well-defined structure and good solubility, such as quantitative labeling and reference standard preparation | |
AF488 | Azide (Click) | AF 488 azide | ≥99% | General-purpose green-channel azide click substrate | Metabolic click staining; protein/carbohydrate tracing | |
AF488 | Pre-labeled protein (Streptavidin) | AF 488 streptavidin conjugate | – | AF488 pre-conjugated to streptavidin; not a “small-molecule reactive dye”; can be directly used with biotin-labeled primary antibodies, oligonucleotides, etc. | Used as “AF488-streptavidin” in ELISA, Western blot, IF, and flow cytometry as a secondary/amplification system | |
AF488 | Alkyne (Click, 5-position) | AF 488 alkyne | – | – | Defined 5-position isomer, providing a more uniform structure | CuAAC with azide substrates; construction of structurally well-defined probes |
AF488 | DBCO (copper-free Click) | AF 488 DBCO | – | For SPAAC copper-free click; good cell compatibility | Copper-free click labeling of azide-containing proteins, nucleic acids, polysaccharides, nanoparticles; suitable for live-cell/animal imaging and molecular tracing | |
AF488 | DBCO (copper-free Click) | AF 488 DBCO, di-triethylamine salt | – | Triethylamine salt form of AF488 DBCO; further improved solubility; functionally equivalent to AF488 DBCO | Same as above; used for copper-free click labeling of azide-modified biomolecules | |
AF488 | Maleimide (C5 spacer arm) | AF 488 C5 Maleimide | – | – | Thiol-reactive dye with a C5 spacer, reducing steric hindrance | Site-specific Cys labeling; protein conformation/dynamics studies |
AF555 | NHS ester (amine-reactive) | AF 555 NHS ester | – | Orange-red AF555 activated ester; good water solubility; forms stable amide bonds with primary amines at pH 7.5–8.5; suitable replacement for Cy3/TAMRA | Brighter and more photostable than traditional dyes after antibody labeling | |
AF555 | Azide (Click) | AF 555 azide | – | Azide derivative of AF555; can undergo efficient CuAAC or SPAAC with alkyne substrates, while retaining the high brightness and good water solubility of AF555 | Click labeling of alkyne-modified antibodies, proteins, peptides, oligonucleotides; live-cell or in vivo tracing; multicolor imaging | |
AF555 | Carboxylic acid | AF 555 carboxylic acid | – | Orange-red AF555 carboxylic acid; extremely hydrophilic; suitable as a reference standard or for preparing activated esters/conjugates | In-house preparation of NHS esters or other derivatives; covalent grafting onto small molecules, nanoparticles, or polymers; fluorescence standard curves | |
AF568 | Carboxylic acid | AF 568 carboxylic acid / AF568 COOH | – | Orange-red channel; high extinction coefficient; stable at pH 4–10 | Preparation of in-house derivatives; multicolor immunofluorescence; FRET donor/acceptor | |
AF568 | NHS ester (amine-reactive) | AF 568 NHS ester | – | Common orange-red dye for antibody/protein labeling; photobleaching-resistant | Confocal imaging, flow cytometry, multiplex IHC/IF | |
AF568 | Alkyne (Click) | AF 568 alkyne | – | Orange-red alkyne for click chemistry | Used with AF568-azide for click reactions; labeling of small molecules/oligonucleotides | |
AF568 | Azide (Click) | AF 568 azide | – | Suitable for projects requiring fine structural definition | Pre-modify carriers via IEDDA and subsequently perform click labeling; metabolic tracing | |
AF568 | Click – DBCO (copper-free) | AF 568 DBCO | – | Orange-red AF568 dye coupled to DBCO strained alkyne; suitable for copper-free SPAAC reactions; good compatibility with cellular and in vivo systems | Copper-free click labeling; live-cell/in vivo imaging; multicolor fluorescent labeling and molecular tracking | |
AF594 | NHS ester (amine-reactive, mixed isomers) | AF 594 NHS ester | ≥95% | Texas Red replacement; red channel; good water solubility | Antibody/protein labeling; multiplex IHC/IF; red channel in flow cytometry | |
AF594 | Click – DBCO (copper-free) | AF 594 DBCO | – | Conjugated to DBCO strained alkyne; suitable for SPAAC copper-free click; soluble in aqueous/organic media; compatible with cellular and in vivo systems | Copper-free click labeling for fluorescence imaging and flow cytometry | |
AF594 | Click – Azide (azide) | AF 594 azide | – | AF 594 azide; red azide click dye; reacts with terminal alkynes via CuAAC | Click labeling of proteins, small molecules, and nanocarriers | |
AF647 | Carboxylic acid | AF 647 carboxylic acid / AF647 acid | – | Far-red carboxylic acid dye; low background; strong penetration | Building AF647 NHS/click derivatives; used as reference standard | |
AF647 | NHS ester (amine-reactive) | AF 647 NHS ester / AF647-NHS ester | – | Cy5 replacement; Ex/Em ~651/672 nm; high extinction coefficient | CAR-T–related flow cytometry, IHC, exploration of antibody–drug conjugates | |
AF647 | Amine | AF 647 amine | – | – | Far-red AF core with amine; can be further activated or conjugated | Reactions with NHS esters, isothiocyanates, etc., for in-house probe preparation |
AF647 | Maleimide (thiol-reactive) | AF 647 Maleimide | – | – | Far-red thiol-specific dye; suitable for low-background, high-sensitivity applications | Site-specific Cys labeling; protein localization; live-cell/long-term imaging |
AF647 | Azide (Click) | AF647 Azide, triethylammonium salt | BioReagent ≥90% (HPLC) | Far-red azide click dye; suitable for both copper and copper-free systems | Metabolic click visualization; in vivo tracing | |
AF647 | Alkyne (Click) | AF 647 alkyne | – | – | Far-red alkyne click dye; for coupling to various azide substrates | Construction of multivalent probes; surface modification |
AF647 | DBCO (copper-free Click) | AF 647 DBCO | – | – | SPAAC copper-free click; highly compatible with cellular/animal systems | In vivo bioorthogonal labeling; construction of ADCs/nanomedicines |
AF647 | Me-Tetrazine (IEDDA Click) | AF 647 Me-Tetrazine | – | – | Ultra-fast IEDDA reaction with TCO/BCN, ideal for in vivo imaging | Pretargeted imaging; in vivo click systems; multistep labeling |
AF660 | NHS ester (amine-reactive) | AF 660 NHS ester | BioReagent ≥90% (HPLC) | Far-red AF660 activated ester; Ex/Em ≈ 668/698 nm; good photostability; low background; suitable for deep tissues and far-red channels in multicolor panels | Far-red labeling of antibodies/proteins; flow cytometry with 633/640 nm laser; multicolor immunophenotyping; tissue section and in vivo imaging | |
AF680 | NHS ester (amine-reactive) | AF 680 NHS ester | – | Near-infrared AF680 activated ester; Ex/Em ≈ 679/702 nm; low autofluorescence interference; strong tissue penetration | NIR labeling of antibodies, protein therapeutics, and nanocarriers; small-animal in vivo imaging; PK and biodistribution studies | |
AF700 | NHS ester (amine-reactive) | AF 700 NHS ester | Ex: 696 nm Em: 719 nm | Near-infrared AF700 activated ester; Ex/Em ≈ 702/723 nm; good photostability; suitable for extending far-red/NIR panels | Far-red/NIR multicolor flow cytometry panels; in vivo or ex vivo tissue imaging; in combination with AF647/680 to build 8–10+ color panels |
Aladdin: https://www.aladdinsci.com/
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