Fluoresceins

Fluorescein derivatives are one of the most commonly used fluorescent labels for biological detection in flow cytometry and immunofluorescnece 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.

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
Figure 1. Excitation and emission spectra for fluorescein-5-isothiocyanate (5-FITC) *CAS 3326-32-7* (Cat No. 121).

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).

Figure 2. Comparison of fluorescence intensity between HeLa cells labeled with GAM IgG-FITC and GAM IgG-iFluor™ 488.
Figure 3. 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 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

Note

  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

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 (-NH?) 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* 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//stratech.tempurl.host/products/113-AAT/ Succinimidyl Ester 493 517 83000 10 mg 113
5-FAM-X, SE *CAS 148356-00-7* Amine Succinimidyl Ester 493 517 83000 5 mg 113
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
6-FAM, SE *CAS 92557-81-8* Amine Succinimidyl Ester 493 517 83000 10 mg 116
6-FAM Azide Alkyne Azide 493 517 83000 10 mg 133
6-FAM Alkyne Azide Alkyne 493 517 83000 10 mg 134
6-FAM *CAS 3301-79-9* Amines after pre-activation with carbodiimides Carboxylic Acid 493 517 83000 100 mg 106
6-FAM phosphoramidite *CAS 204697-37-0* Oligonucleotide 5’ end labeling Phosphoramidite 493 517 83000 100 µmoles 6016
OG488 maleimide Thiol Maleimide 498 526 76000 5 mg 715
5-OG488 succinimidyl ester Amine Succinimidyl Ester 498 526 76000 5 mg 710
5-OG488 carboxylic acid Amines after pre-activation with carbodiimides Carboxylic Acid 498 526 76000 25 mg 708
6-OG488 succinimidyl ester Amine Succinimidyl Ester 498 526 76000 5 mg 711
6-OG488 carboxylic acid Amines after pre-activation with carbodiimides Carboxylic Acid 498 526 76000 25 mg 712
6-TET, SE Amine Succinimidyl Ester 521 542 73000 5 mg 211
6-TET azide Alkyne Azide 521 542 73000 5 mg 244
6-TET alkyne Azide Alkyne 521 542 73000 5 mg 245
6-TET phosphoramidite *CAS 877049-90-6* Oligonucleotide 5’ end labeling Phosphoramidite 521 542 73000 50 µmoles 6021
6-JOE, SE Amine Succinimidyl Ester 520 545 75000 5 mg 203
6-JOE, SE Amine Succinimidyl Ester 520 545 75000 5 mg 203
6-JOE azide Alkyne Azide 520 545 75000 5 mg 248
6-JOE Phosphoramidite Oligonucleotide 5’ end labeling Phosphoramidite 520 545 75000 100 µmoles 6047
6-HEX, SE *Single Isomer* Amine Succinimidyl Ester 533 559 73000 5 mg 202
6-HEX azide Alkyne Azide 533 559 73000 5 mg 140
6-HEX alkyne Azide Alkyne 533 559 73000 5 mg 241
6-HEX phosphoramidite Oligonucleotide 5’ end labeling Phosphoramidite 533 559 73000 100 µmoles 6026
FITC-xtra Amine Succinimidyl Ester 498 526 73000 5 mg 135
5-FITC isothiocyanate *CAS 3326-32-7* Amine Isothiocyanate 491 516 73000 1 g 121
5-IAF [5-Iodoacetamidofluorescein] Thiol Iodoacetamide 498 517 80000 25 mg 222
Fluorescein-5-maleimide Thiol Maleimide 498 517 80000 25 mg 130
5-FITC cadaverine Labels proteins by transaminidation Cadaverine 491 516 73000 100 mg 129
5-FITC ethylenediamine Aldehyde, Carboxylic Acid, Ketone Ethylenediamine 491 516 73000 100 mg 129
6-Fluorescein phosphoramidite Oligonucleotide 5’ end labeling Phosphoramidite 498 517 80000 100 µmoles 6018
3′-Fluorescein CPG *1000 Å* Oligonucleotide 3’ end labeling 487 517 80000 100 µmoles 6011
3′-(6-Fluorescein) CPG Oligonucleotide 3’ end labeling 487 517 80000 1 g 6014
5-DTAF *CAS 51306-35-5* Amines and Thiols Dichlorotriazine 498 517 80000 25 mg 200
AMF [4′-(Aminomethyl)fluorescein] Activated Carboxy Esters (NHS Esters) Amino 487 517 80000 25 mg 206
Tide Fluor™ 2 succinimidyl ester Amine Succinimidyl Ester 503 525 75000 5 mg 2248
Tide Fluor™ 2 maleimide Thiol Maleimide 503 525 75000 1 mg 2247
Tide Fluor™ 2 carboxylic acid Amines after pre-activation with carbodiimides Carboxylic Acid 503 525 75000 25 mg 2245
Tide Fluor™ 2 azide Alkyne Azide 503 525 75000 1 mg 2252
Tide Fluor™ 2 amine Carbonyls Amine 503 525 75000 1 mg 2246
Tide Fluor™ 2 alkyne Azide Alkyne 503 525 75000 5 mg 2253
Tide Fluor™ 2WS succinimidyl ester Amine Succinimidyl Ester 503 525 75000 5 mg 2349
Tide Fluor™ 2WS maleimide Thiol Maleimide 503 525 75000 1 mg 2350
Tide Fluor™ 2WS carboxylic acid Amines after pre-activation with carbodiimides Carboxylic Acid 503 525 75000 10 mg 2348
Tide Fluor™ 2WS amine Carbonyls Amine 503 525 75000 1 mg 2351

Note

  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 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
Glucose-UDP-(PEG)6-Fluorescein Conjugate Glucosyltransferase Substrate 498 517 100 µg 11706

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 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
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
FITC–dextran conjugate (average MW = ~20K) Cardiovascular, Microcirculation, Perfusion, Cell Membrane Flux Tracer 491 516 25 mg 21700
FITC–dextran conjugate (average MW = ~70K) Cardiovascular, Microcirculation, Perfusion, Cell Membrane Flux Tracer 491 516 25 mg 21702
FITC–dextran conjugate (average MW = ~150K) Cardiovascular, Microcirculation, Perfusion, Cell Membrane Flux Tracer 491 516 25 mg 21704

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 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
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
BCECF, AM Cell-Permeable pH Indicator 504 527 1 mg 21202
BCECF acid *CAS#: 85138-49-4* pH Indicator 504 527 1 mg 21201
CytoFix™ BCECF, AM *Optimized for long term cellular pH tracking* Cell-Permeable and Fixable pH Indicator 504 527 1 mg 21200
RatioWorks™ BCFL, AM Cell-Permeable pH Indicator 503 524 1 mg 21190
Fluo-2, AM Cell-Permeable Calcium Indicator (Kd = 232 nM) 488 515 1 mg 20495
Fluo-2, potassium salt Cell-Impermeable Calcium Indicator (Kd = 232 nM) 488 515 10 x 50 µg 20493
Fluo-3, AM *UltraPure grade* *CAS 121714-22-5* Cell-Permeable Calcium Indicator (Kd = 390 nM) 507 516 1 mg 21011
Fluo-3, pentapotassium salt Cell-Impermeable Calcium Indicator (Kd = 390 nM) 507 516 1 mg 21017
Fluo-3, pentaammonium salt Cell-Impermeable Calcium Indicator (Kd = 390 nM) 507 516 1 mg 21017
Fluo-3, pentasodium salt Cell-Impermeable Calcium Indicator (Kd = 390 nM) 507 516 1 mg 21016
Fluo-4 AM *Ultrapure Grade* *CAS 273221-67-3* Cell-Permeable Calcium Indicator (Kd = 345 nM) 495 528 1 mg 20550
Fluo-4, Pentapotassium Salt Cell-Impermeable Calcium Indicator (Kd = 345 nM) 495 528 1 mg 20555
Mag-Fluo-4 AM Cell-Permeable Mg Indicator (Kd = 4.7 mM), Low-Affinity Calcium Indicator (Kd = 22 µM) 495 528 10 x 50 µg 20401
Mag-Fluo-4 potassium salt Cell-Permeable Mg Indicator (Kd = 4.7 mM), Low-Affinity Calcium Indicator (Kd = 22 µM) 495 528 10 x 50 µg 20400
Fluo-5F, AM Cell-Permeable Calcium Indicator (Kd = 2.3 µM) 494 516 10 x 50 µg 20560
Fluo-5F, pentapotassium Salt Cell-Impermeable Calcium Indicator (Kd = 2.3 µM) 494 516 500 µg 20562
Fluo-5N, AM Cell-Permeable Calcium Indicator (Kd = 90 µM) 494 516 10 x 50 µg 20566
Fluo-5N, pentapotassium Salt Cell-Impermeable Calcium Indicator (Kd = 90 µM) 494 516 500 µg 20567
Fluo-8®, AM Cell-Permeable Calcium Indicator (Kd = 389 nM) 495 516 1 mg 21080
Fluo-8®, potassium salt Cell-Impermeable Calcium Indicator (Kd = 389 nM) 495 516 1 mg 21087
Fluo-8®, sodium salt Cell-Impermeable Calcium Indicator (Kd = 389 nM) 495 516 1 mg 21086
Fluo-8H™, AM Cell-Impermeable Calcium Indicator (Kd = 232 nM) 495 516 1 mg 21090
Fluo-8H™, sodium salt Cell-Impermeable Calcium Indicator (Kd = 232 nM) 495 516 10 x 50 µg 21095
Fluo-8FF™, AM Cell-Impermeable Calcium Indicator (Kd = 10 µM) 495 516 1 mg 21105
Fluo-8FF™, potassium salt Cell-Impermeable Calcium Indicator (Kd = 10 µM) 495 516 1 mg 21103
Fluo-8L™, AM Cell-Impermeable Calcium Indicator (Kd = 1.9 µM) 495 516 1 mg 21096
Fluo-8L™, sodium salt Cell-Impermeable Calcium Indicator (Kd = 1.9 µM) 495 516 1 mg 21099
Fluo-8L™, potassium salt Cell-Impermeable Calcium Indicator (Kd = 1.9 µM) 495 516 1 mg 21101

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