Fluorescein derivatives are one of the most commonly used fluorescent labels for biological detection in flow cytometry and immunofluorescence due to their high absorptivity, excellent fluorescence quantum yields and good water solubility. The most widely used fluoresceins include fluorescein isothiocyanate (FITC)
for labeling proteins (in particular antibodies) and carboxyfluoresceins (5-FAM
) for labeling peptides
and oligonucleotides. We offer a broad selection of fluorescein based-dyes, substrates and conjugates, as well as a series of superior iFluor® fluorescent labeling dyes
with improved characteristics optimized for cellular labeling and detection.
Excitation and emission spectra for fluorescein-5-isothiocyanate (5-FITC) *CAS 3326-32-7* (Cat No. 121
Table 1. Spectral properties of fluorescein-5-isothiocyanate (5-FITC).
|Laser Line:||488 nm laser|
|Ex/Em (nm):||494/525 nm|
|Extinction Coefficient (ε):||75,000 cm-1M-1|
|Quantum Yield (Φ):||0.79|
iFluor® 488 - superior alternative to FITC & Alexa Fluor® 488
Despite its bright green fluorescence, fluorescein based-dyes and conjugates have a number of caveats that may restrict its use in certain applications, including rapid photobleaching, pH-sensitive fluorescence and susceptibility to quenching. The iFluor® 488 dye-with identical spectral properties - produces brighter, more photostable conjugates with pH-insensitive fluorescence over a wide molar range. iFluor® 488 dye molecules can be conjugated to biomolecules at high molar ratios with minimal self-quenching (Figure 2 and Figure 3). Learn More
Comparison of fluorescence intensity between HeLa cells labeled with GAM IgG-FITC and GAM IgG-iFluor® 488. Comparison of photobleaching rates of iFluor® 488 goat anti-mouse IgG and the conventional fluorescein (FITC) goat anti-mouse IgG. Tubulin of HeLa cells was labeled with (top series) iFluor® 488 GAM-IgG or (bottom series) FITC GAM-IgG. Images were taken at 60-second intervals on a Keyence X710 fluorescence microscope. TFI represents total fluorescence intensity as calculated by ImageJ software.
Table 2. Available iFluor® 488 Reactive Dyes for Labeling Biomolecules.
|iFluor® 488 succinimidyl ester||Amine||Succinimidyl Ester||491||516||75000||1 mg||1023|
|iFluor® 488 maleimide||Thiol||Maleimide||491||516||75000||1 mg||1062|
|iFluor® 488 alkyne||Azide||Alkyne||491||516||75000||1 mg||999|
|iFluor® 488 amine||Aldehyde, Carboxylic Acid, Ketone||Amine||491||516||75000||1 mg||1072|
|iFluor® 488 azide||Alkyne||Azide||491||516||75000||1 mg||1000|
|iFluor® 488 hydrazide||Aldehyde, Carboxylic Acid, Ketone||Hydrazide||491||516||75000||1 mg||1082|
- ε = molar extinction coefficient at their maximum absorption wavelength (Units = cm-1M-1).
We offer a broad selection of fluorescein products including reactive fluorescein and fluorescein derivatives for labeling antibodies, nucleic acids and other biomolecules, conjugates, indicators, and substrates for detecting enzymatic activity in cells, homogenates and solution. Learn More
Table 3. Fluorescein Reference Standards
Table 4. Fluorescein Precursors
Table 5. Fluorescein Calibrators
Table 6. FRET Building Blocks
Table 7. DNA Sequencing Building Blocks
|300||5-dR6G [5-Carboxy-4,7-dichlororhodamine 6G]||25 mg|
|301||6-dR6G [6-Carboxy-4,7-dichlororhodamine 6G]||25 mg|
|302||5-dR6G, succinimidyl ester||5 mg|
|305||5-dTMR [5-Carboxy-4,7-dichlorortetramethylrhodamine]||25 mg|
|306||6-dTMR [6-Carboxy-4,7-dichlorortetramethylrhodamine]||25 mg|
|307||5-dTMR, succinimidyl ester||5 mg|
|310||5-dROX [5-Carboxy-4,7-dichloror-X-hodamine]||25 mg|
|311||6-dROX [6-Carboxy-4,7-dichloror-X-hodamine]||25 mg|
|312||5-dROX, succinimidyl ester||5 mg|
|315||5-dR110 [5-Carboxy-4,7-dichlororhodamine 110]||25 mg|
Fluorescein Reactive Dyes
Reactive fluorescent fluorescein and fluorescein derivatives are widely used to covalently label biomolecules such as antibodies, carbohydrates, nucleic acids, proteins, peptides and oligonucleotides. Resulting in intensely bright and photostable green fluorescence, fluorescein conjugates are often used as substrates for detecting enzymatic activity or to provide contrast in multicolored fluorescence investigation when combined with blue, yellow or red fluorescent probes. Fluorescein and fluorescein derivatives are readily available in a variety of dye-labeling chemistries, these include:
- Succinimidyl esters/NHS esters - for labeling free amines (-NH2) on antibodies, proteins, peptides, amine-modified oligonucleotides and other biomolecules
- Maleimides - for labeling sulfhydryl groups on antibodies, proteins and peptides
- Azides - for labeling ethylene groups via click chemistry methods
- Alkynes - for labeling azides via click chemistry methods
- Hydrazides - for labeling aldehyde and ketone groups
- Carboxylic acids - for labeling amines after pre-activation with carbodiimides or for Steglich esterification of alcohols
- Amines - for labeling with various electrophilic compounds such as activated esters
- Tetrazines - for labeling trans-cyclooctenes (TCO) compounds via Inverse-Electron-Demands Diels-Alder reaction
- Phosphoramidite - for labeling oligonucleotides
Table 8. Available Reactive Fluorescein and Fluorescein Derivatives for Labeling Biomolecules.
|5(6)-FAM, SE *CAS 117548-22-8*||Amine||Succinimidyl Ester||493||517||83000||25 mg||110|
|5(6)-FAM *CAS 72088-94-9*||Amines after pre-activation with carbodiimides||Carboxylic Acid||493||517||83000||1 g||100|
|5(6)-FAM ethylenediamine||Aldehyde, Carboxylic Acid, Ketone||Ethylenediamine||493||517||83000||100 mg||123|
|5-FAM, SE||Amine||Succinimidyl Ester||493||517||83000||10 mg||113|
|5-SFX [5-FAM-X, SE]||Amine||Succinimidyl Ester||493||517||83000||5 mg||119|
|5-FAM cadaverine||Labels proteins by transaminidation||Cadaverine||493||517||83000||100 mg||128|
|5-FAM Azide||Alkyne||Azide||493||517||83000||10 mg||131|
|5-FAM Alkyne||Azide||Alkyne||493||517||83000||10 mg||132|
|5-FAM *CAS 76823-03-5*||Amines after pre-activation with carbodiimides||Carboxylic Acid||493||517||83000||100 mg||103|
|5-FAM ethylenediamine||Aldehyde, Carboxylic Acid, Ketone||Ethylenediamine||493||517||83000||100 mg||124|
- ε = molar extinction coefficient at their maximum absorption wavelength (Units = cm-1M-1).
Fluorescein-Labeled Enzyme Substrates
Fluorescein and fluorescein derivatives are frequently used to prepare fluorogenic substrates for detecting enzymatic activity in cells and solutions. When hydrolyzed by the enzyme of interest, fluorescein-modified substrates exhibit an increase in fluorescence intensity that is linearly proportional to enzyme activity. AAT Bioquest provides a large assortment of fluorescein modified substrates for detecting activity from caspases, proteases, peptidases, phosphatases and more.
Table 9. Available Fluorescein-Labeled Fluorogenic Substrates.
|FDG [Fluorescein di-beta-D-galactopyranoside]||β-galactosidase substrate||498||517||1 g||14001|
|FDP [Fluorescein diphosphate, tetraammonium salt] *CAS 217305-49-2*||Phosphatase Substrate||498||517||5 mg||11600|
|FCB [Fluorescein di-beta-D-cellobioside]||Cellulase (e.g. β-glucosidases, endoglucanases and exoglucanases) Substrate||498||517||1 mg||14025|
|Casein, FITC-conjugated||Generic Protease Substrate||498||517||5 mg||13440|
|FAM-VAD-FMK||Polycaspase Inhibitor (Caspases 1, 2, 3, 6, 8, 9, or 10)||493||517||25 Tests||13470|
|FITC-C6-DEVD-FMK||Caspase 3 Substrate||491||516||100 µg||13408|
|FITC-C6-D(OMe)E(OMe)VD(OMe)-FMK||Caspase 3 Substrate||491||516||100 µg||13417|
|FITC-C6-LEHD-FMK||Caspase 9 Substrate||491||516||100 µg||13409|
|FDGlcU [Fluorescein di-beta-D-glucuronide]||β-Glucuronidase Substrate||498||517||1 mg||14002|
|Glucose-UDP-Fluorescein Conjugate||Glucosyltransferase Substrate||498||517||100 µg||11705|
Fluorescein-labeled conjugates have a wide range of applications in bio-imaging and bio-labelling due to their bright green fluorescence and excellent photostability. Fluorescein derivatives can be labeled to antibodies, streptavidin, phalloidin, annexin v, dextran and other biomolecules to study apoptosis, label organelles or improve assay sensitivity by amplifying weak signals.
Table 10. Available Fluorescein-Labeled Fluorogenic Conjugates.
|Biotin-4-fluorescein *CAS 1032732-74-3*||Binds with high-affinity to streptavidin tetramers||492||518||5 mg||3006|
|Annexin V, FITC Labeled||Detects phosphatidylserine on the cell surface||491||516||100 Tests||20030|
|Phalloidin-Fluorescein Conjugate||Labels actin filaments (F-actin)||498||517||300 Tests||23101|
|Fluorescein DHPE||Measuring lateral diffusion in membranes||498||517||5 mg||23304|
|FITC-streptavidin conjugate||Indirectly binds to biotinylated primary antibodies, amplifies signal||491||516||1 mg||16910|
|Fluorescein biotin||Binds with high-affinity to streptavidin tetramers||498||517||5 mg||3017|
|FITC goat anti-mouse IgG (H+L)||Indirectly detects mouse primary antibodies||491||516||1 mg||16852|
|FITC goat anti-mouse IgG (H+L) *Cross Adsorbed*||Indirectly detects mouse primary antibodies||491||516||1 mg||16860|
|FITC goat anti-rabbit IgG (H+L)||Indirectly detects rabbit primary antibodies||491||516||1 mg||16868|
|FITC goat anti-rabbit IgG (H+L) *Cross Adsorbed*||Indirectly detects rabbit primary antibodies||491||516||1 mg||16876|
Fluorescein-derived indicators are important tools in biochemical and cellular studies. They have been used in all major types of fluorescence imaging applications including flow cytometry, fluorescence microscopy and fluorescence plate-based assays. AAT Bioquest offers a wide range of fluorescein-derived indicators for measuring cellular pH, calcium concentrations, cell proliferation, cell viability and more.
Table 11. Available Fluorescein-Derived Indicators.
|FDA [Fluorescein diacetate] *CAS 596-09-8*||Cell Viability Indicator||498||517||1 g||22020|
|Fluorescein dicaprylate [Fluorescein dioctanoate]||Cell Viability Indicator||498||517||100 mg||22019|
|Fluorescein dicaprylate *Water-Soluble*||Cell Viability Indicator||498||517||5 mg||22018|
|CFSE [5-(and 6)-Carboxyfluorescein diacetate, succinimidyl ester] *CAS 150347-59-4*||Cell Proliferation Indicator, Cell Viability Indicator||498||517||25 mg||22022|
|CDCF [5-(and-6)-Carboxy-2',7'-dichlorofluorescein] *Mixed isomers*||ROS Indicator||505||526||100 mg||235|
|CDCFDA, SE [5-(and-6)-Carboxy-2',7'-dichlorofluorescein diacetate, succinimidyl ester] *Mixed isomers*||Cell Proliferation Indicator, Cell Viability Indicator||498||517||25 mg||22026|
|ReadiUse™ CFSE [5-(and 6)-Carboxyfluorescein diacetate, succinimidyl ester] *CAS 150347-59-4*||Cell Proliferation Indicator, Cell Viability Indicator||498||517||5 x 500 µg||22028|
|CDCFDA [5-(and-6)-Carboxy-2',7'-dichlorofluorescein diacetate] *Mixed isomers*||Cell Viability Indicator||498||517||100 mg||22025|
|CFDA [5-(and 6)-Carboxyfluorescein diacetate] *Mixed isomers*||Cell Viability Indicator||498||517||100 mg||22021|
|Dihydrofluorescein diacetate [Fluorescin diacetate] *CAS 35340-49-9*||Cell Viability Indicator, Redox Indicator||498||517||100 mg||15203|