Actively helping customers, employees and the global community during the coronavirus SARS-CoV-2 outbreak.  Learn more >>

Fluoresceins

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 and 5(6)-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.

 

 

Spectral Properties



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).

Specification
Value
Laser Line:488 nm laser
Filter Set:FITC
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.

Product
Chemical Reactivity
Reactive Moiety
Ex (nm)
Em (nm)
ε1
Unit Size
Cat No.
iFluor® 488 succinimidyl esterAmineSuccinimidyl Ester491516750001 mg1023
iFluor® 488 maleimideThiolMaleimide491516750001 mg1062
iFluor® 488 alkyneAzideAlkyne491516750001 mg999
iFluor® 488 amineAldehyde, Carboxylic Acid, KetoneAmine491516750001 mg1072
iFluor® 488 azideAlkyneAzide491516750001 mg1000
iFluor® 488 hydrazideAldehyde, Carboxylic Acid, KetoneHydrazide491516750001 mg1082
  1. ε = molar extinction coefficient at their maximum absorption wavelength (Units = cm-1M-1).

 

Fluorescein Products


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.

FRET Building-Blocks


DNA Sequencing Building-Blocks



Learn More

 

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 3. Available Reactive Fluorescein and Fluorescein Derivatives for Labeling Biomolecules.

Product
Chemical Reactivity
Reactive Moiety
Ex (nm)
Em (nm)
ε1
Unit Size
Cat No.
5(6)-FAM, SE *CAS 117548-22-8*AmineSuccinimidyl Ester4935178300025 mg110
5(6)-FAM *CAS 72088-94-9*Amines after pre-activation with carbodiimidesCarboxylic Acid493517830001 g100
5(6)-FAM ethylenediamineAldehyde, Carboxylic Acid, KetoneEthylenediamine49351783000100 mg123
5-FAM, SEAmineSuccinimidyl Ester4935178300010 mg113
5-FAM-X, SE *CAS 148356-00-7*AmineSuccinimidyl Ester493517830005 mg113
5-FAM AzideAlkyneAzide4935178300010 mg131
5-FAM AlkyneAzideAlkyne4935178300010 mg132
5-FAM *CAS 76823-03-5*Amines after pre-activation with carbodiimidesCarboxylic Acid49351783000100 mg103
5-FAM ethylenediamineAldehyde, Carboxylic Acid, KetoneEthylenediamine49351783000100 mg124
6-FAM, SE *CAS 92557-81-8*AmineSuccinimidyl Ester4935178300010 mg116
  1. 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 4. Available Fluorescein-Labeled Fluorogenic Substrates.

Product
Biological Function
Ex (nm)
Em (nm)
Unit Size
Cat No.
FDG [Fluorescein di-beta-D-galactopyranoside]β-galactosidase substrate4985171 g14001
FDP [Fluorescein diphosphate, tetraammonium salt] *CAS 217305-49-2*Phosphatase Substrate4985175 mg11600
FCB [Fluorescein di-beta-D-cellobioside]Cellulase (e.g. β-glucosidases, endoglucanases and exoglucanases) Substrate4985171 mg14025
Casein, FITC-conjugatedGeneric Protease Substrate4985175 mg13440
FAM-VAD-FMKPolycaspase Inhibitor (Caspases 1, 2, 3, 6, 8, 9, or 10)49351725 Tests13470
FITC-C6-DEVD-FMKCaspase 3 Substrate491516100 µg13408
FITC-C6-D(OMe)E(OMe)VD(OMe)-FMKCaspase 3 Substrate491516100 µg13417
FITC-C6-LEHD-FMKCaspase 9 Substrate491516100 µg13409
FDGlcU [Fluorescein di-beta-D-glucuronide]β-Glucuronidase Substrate4985171 mg14002
Glucose-UDP-Fluorescein ConjugateGlucosyltransferase Substrate498517100 µg11705

 

Fluorescein-Labeled Conjugates


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 5. Available Fluorescein-Labeled Fluorogenic Conjugates.

Product
Biological Function
Ex (nm)
Em (nm)
Unit Size
Cat No.
Biotin-4-fluorescein *CAS 1032732-74-3*Binds with high-affinity to streptavidin tetramers4925185 mg3006
Annexin V, FITC LabeledDetects phosphatidylserine on the cell surface491516100 Tests20030
Phalloidin-Fluorescein ConjugateLabels actin filaments (F-actin)498517300 Tests23101
FITC-streptavidin conjugateIndirectly binds to biotinylated primary antibodies, amplifies signal4915161 mg16910
Fluorescein biotinBinds with high-affinity to streptavidin tetramers4985175 mg3017
FITC goat anti-mouse IgG (H+L)Indirectly detects mouse primary antibodies4915161 mg16852
FITC goat anti-mouse IgG (H+L) *Cross Adsorbed*Indirectly detects mouse primary antibodies4915161 mg16860
FITC goat anti-rabbit IgG (H+L)Indirectly detects rabbit primary antibodies4915161 mg16868
FITC goat anti-rabbit IgG (H+L) *Cross Adsorbed*Indirectly detects rabbit primary antibodies4915161 mg16876
FITC–dextran conjugate (average MW = ~20K)Cardiovascular, Microcirculation, Perfusion, Cell Membrane Flux Tracer49151625 mg21700

 

Fluorescein-Derived Indicators


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 6. Available Fluorescein-Derived Indicators.

Product
Biological Function
Ex (nm)
Em (nm)
Unit Size
Cat No.
FDA [Fluorescein diacetate] *CAS 596-09-8*Cell Viability Indicator4985171 g22020
Fluorescein dicaprylate [Fluorescein dioctanoate]Cell Viability Indicator498517100 mg22019
Fluorescein dicaprylate *Water-Soluble*Cell Viability Indicator4985175 mg22018
CFSE [5-(and 6)-Carboxyfluorescein diacetate, succinimidyl ester] *CAS 150347-59-4*Cell Proliferation Indicator, Cell Viability Indicator49851725 mg22022
CDCFDA, SE [5-(and-6)-Carboxy-2',7'-dichlorofluorescein diacetate, succinimidyl ester] *Mixed isomers*Cell Proliferation Indicator, Cell Viability Indicator49851725 mg22026
ReadiUse™ CFSE [5-(and 6)-Carboxyfluorescein diacetate, succinimidyl ester] *CAS 150347-59-4*Cell Proliferation Indicator, Cell Viability Indicator4985175 x 500 µg22028
CDCFDA [5-(and-6)-Carboxy-2',7'-dichlorofluorescein diacetate] *Mixed isomers*Cell Viability Indicator498517100 mg22025
CFDA [5-(and 6)-Carboxyfluorescein diacetate] *Mixed isomers*Cell Viability Indicator498517100 mg22021
Dihydrofluorescein diacetate [Fluorescin diacetate] *CAS 35340-49-9*Cell Viability Indicator, Redox Indicator498517100 mg15203
BCECF, AMCell-Permeable pH Indicator5045271 mg21202