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AAT Bioquest

Coumarins

Coumarin and coumarin derivatives are UV-excitable blue fluorescent dyes with short emission wavelengths ranging from ~390 to ~480 nm. The most widely used coumarins include AMCA, the derivatives of 7-aminocoumarin (AFC and AMC), 7-hydoxycoumarin and 7-methoxycoumarin for preparing blue fluorescent peptides, proteins and other biomolecules. Compared to fluoresceins, rhodamines, and cyanines, coumarins produce moderately bright and photostable fluorescence due to their relatively low extinction coefficients.

Blue-fluorescent probes are best used for direct imaging of high-abundant targets such as cytoskeleton markers, in cell tracking applications, as substrates for detecting enzyme activity and for certain multicolor fluorescence applications - including immunofluorescence, nucleic acid and protein microarrays, in situ hybridization and neuronal tracing.

 

 

Spectral Properties


AMCA spectra
Excitation and emission spectra of AMCA, a commonly used coumarin derivative.
 

Table 1. Spectral properties of 7-Amino-4-trifluoromethylcoumarin.

Specification
Value
Laser Line:405 nm laser
Filter Set:DAPI
Ex/Em (nm):376/480
Extinction Coefficient (ε):17,000 cm-1M-1
Quantum Yield (Φ):0.53



 

iFluor® 350 - a superior alternative to AMCA and Alexa Fluor® 350


HeLa cells were stained with Phalloidin ifluor 350 conjugate
HeLa cells were fixed in 4% formaldehyde and then stained with Phalloidin iFluor® 350 conjugate and Nuclear Orange™ DCS1.
The iFluor® 350 dye is a bright blue fluorescent dye spectrally similar to AMCA with improved water-solubility, photostability and higher fluorescence quantum yields. iFluor® 350 conjugates are well-suited for direct imaging of high abundance targets, and is particularly useful in multicolor applications, such as flow cytometry and super-resolution fluorescence microscopy, without compromising the performance of green and red fluorescent probes.

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Table 2. iFluor® 350 - a superior alternative to AMCA and Alexa Fluor® 350

Fluorophore
Quantum Yield
Extinction Coefficient
Brightness
iFluor® 3500.9520,000**
AMCA0.5319,000*

 

Coumarin Products


We offer a broad selection of coumarin products including reactive dyes for labeling peptides, proteins and other biomolecules, conjugates, indicators and substrates for detecting enzymatic activity in cells, homogenates and solution.

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Coumarin Reactive Dyes


Reactive fluorescent coumarin and coumarin derivatives are widely used to covalently label biomolecules such as antibodies, carbohydrates, nucleic acids, proteins, peptides and oligonucleotides. Resulting in moderately bright and photostable blue fluorescence, coumarin conjugates are often used as substrates for detecting enzymatic activity or to provide contrast in multicolored fluorescence investigation when combined with green, yellow or red fluorescent probes. Coumarin and coumarin 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
 

Table 5. Available Reactive Coumarin and Coumarin Derivatives.

Product
Chemical Reactivity
Reactive Moiety
Ex (nm)
Em (nm)
ε1
Unit Size
Cat No.
AMCA acid 3464341900025 mg501
AMCA, succinimidyl esterAmineSuccinimidyl Ester3464341900010 mg502
AMCA azideAlkyneAzide346434190001 mg508
AMCA alkyneAzideAlkyne346434190001 mg507
AMCA C2 maleimideThiolMaleimide346434190005 mg503
AMCA ethylenediamineAldehyde, Carboxylic Acid, KetoneEthylenediamine346434190005 mg504
DEACAmine (after pre-activation with carbodiimides)Carboxylic Acid4114724000025 mg505
DEAC, succinimidyl esterAmineSuccinimidyl Ester4114724000025 mg506
7-Hydroxy-4-methylcoumarin-3-acetic acid 36445825000100 mg554
7-Hydroxy-4-methylcoumarin-3-acetic acid, succinimidyl esterAmineSuccinimidyl Ester3644582500025 mg556
7-Hydroxycoumarin-3-carboxylic acidAmine (after pre-activation with carbodiimides)Carboxylic Acid3524072500025 mg549
7-Hydroxycoumarin-3-carboxylic acidAmine (after pre-activation with carbodiimides)Carboxylic Acid35240725000250 mg550
7-Hydroxycoumarin-3-carboxylic acid, succinimidyl esterAmineSuccinimidyl Ester3524073500050 mg551
7-Hydroxycoumarin-4-acetic acid 36045018000100 mg552
7-Hydroxycoumarin-4-acetic acid, succinimidyl esterAmineSuccinimidyl Ester3604501800025 mg553
MCA [7-Methoxycoumarin-4-acetic acid] *CAS 62935-72-2* 322381 1 g557
MCA succinimidyl esterAmineSuccinimidyl Ester3223811500025 mg558
7-Methoxycoumarin-3-carboxylic acidAmine (after pre-activation with carbodiimides)Carboxylic Acid355405200001 g560
7-Methoxycoumarin-3-carboxylic acidAmine (after pre-activation with carbodiimides)Carboxylic Acid355405200005 g561
7-Methoxycoumarin-3-carboxylic acid, succinimidyl esterAmineSuccinimidyl Ester35540525000100 mg563
iFluor® 350 amineAldehyde, Carboxylic Acid, KetoneAmine345450190001 mg1070
iFluor® 350 hydrazideAldehyde, Carboxylic Acid, KetoneHydrazide345450200001 mg1080
iFluor® 350 maleimideThiolMaleimide343441190001 mg1060
iFluor® 350 succinimidyl esterAmineSuccinimidyl Ester345450200001 mg1020
iFluor® 405 amineAldehyde, Carboxylic Acid, KetoneAmine403427370001 mg1071
iFluor® 405 hydrazideAldehyde, Carboxylic Acid, KetoneHydrazide403427370001 mg1081
iFluor® 405 maleimideThiolMaleimide403427370001 mg1053
iFluor® 405 succinimidyl esterAmineSuccinimidyl Ester403427370001 mg1021
Alpha Fluor™ 350 NHS esterAmineNHS Ester343441190001 mg1800
Alpha Fluor™ 350 C5 maleimideThiolMaleimide343441190001 mg1870
CEC [3-Cyano-7-ethoxycoumarin] *CAS 117620-77-6* 4064504300010 mg15001
  1. ε = molar extinction coefficient at their maximum absorption wavelength (Units = cm-1M-1).

 

Coumarin-Labeled Enzyme Substrates


Coumarin and coumarin derivatives are frequently used to prepare fluorogenic substrates for detecting enzymatic activity in cells and solutions. When hydrolyzed by the enzyme of interest, coumarin-modified substrates exhibit an increase in fluorescence intensity that is linearly proportional to enzyme activity. AAT Bioquest provides a large assortment of coumarin modified substrates for detecting activity from caspases, proteases, peptidases, phosphatases and more.
 

Table 6. Available Coumarin-Labeled Fluorogenic Substrates.

Product
Biological Function
Ex (nm)
Em (nm)
Unit Size
Cat No.
Ac-DEVD-AFC *CAS 201608-14-2*Caspase 3/7 Substrate3764825 mg13401
Ac-DEVD-AMC *CAS 169332-61-0*Caspase 3/7 Substrate3414415 mg13402
Z-DEVD-AFCCaspase 3/7 Substrate3764825 mg13420
Z-DEVD-AMCCaspase 3/7 Substrate3414415 mg13421
Ac-IETD-AFC *CAS 211990-57-7*Caspase 8 Substrate3764825 mg13410
Ac-IETD-AMCCaspase 8 Substrate3414415 mg13411
Z-IETD-AFC *CAS 219138-02-0*Caspase 8 Substrate3764825 mg13425
Ac-LEHD-AMC *CAS 292633-16-0*Caspase 9 Substrate3414415 mg13426
Gly-Pro-AMC *CAS 115035-46-6*Dipeptidyl Peptidase IV (DPPIV) Substrate3414415 mg13450
Ala-AMCAlanyl Aminopeptidase and Trypsin Substrate3414415 mg13458
Ac-Pro-Ala-Leu-AMCProteasome Substrate (26S proteasome)3414415 mg13479
MeO-Succ-Arg-Pro-Tyr-AMCChymotrypsin-like Protease Substrate3414415 mg13460
Suc-LLVY-AMC20S proteasome, Calpains and Chymotrypsin-like Protease Substrate3414415 mg13453
D-VLK-AMC [D-Val-Leu-Lys-AMC]Plasmin Substrate3414415 mg13201
BOC-Val-Pro-Arg-AMCAlpha-Thrombin Substrate3414415 mg13461
Leu-AMC *CAS 62480-44-8*Leucine Aminopeptidase Substrate3414415 mg13445
Z-Gly-Pro-AMC *CAS 68542-93-8*Prolyl Endopeptidase Substrate3414415 mg13477
Gly-Pro-AMC *CAS 115035-46-6*Dipeptidyl Peptidase IV (DPPIV) Substrate3414415 mg13450
D-Ala-AMC *CAS 201847-52-1*Aminopeptidase and Endopeptidase Substrate3414415 mg13459
Benzoyl-Arg-AMCHuman Alpha-Thrombin Substrate3414415 mg 
DiFMUPAlkaline Phosphatase Substrate3414415 mg11627
MUP *CAS 3368-04-5*Phosphatase Substrate (in solution)3414415 mg11614
MUP, disodium salt *CAS 22919-26-2*Phosphatase Substrate (in solution)3414415 mg11610
MUG [4-Methylumbelliferyl-beta-D-galactopyranoside] *CAS 6160-78-7*Galactosidase Substrate34144125 mg14010
4-Methylumbelliferyl-beta-D-glucoside *CAS 18997-57-4*Glucosidase Substrate34144125 mg14014
MUGlcU [4-Methylumbelliferyl-beta-D-glucuronide] *CAS 881005-91-0*Glucuronidase Substrate3414415 mg14016
7-Ethoxy-4-trifluoromethylcoumarin *CAS 115453-82-2*Cytochrome P450 oxidase 25 mg15005
Mca-APK(Dnp) ACE2 substrateACE2 substrate3223811 mg13555
Mca-PLAQAV-Dpa-RSSSR-NH2Tumor necrosis factor-alpha converting enzyme (TACE or ADAM17) substrate3223811 mg13560
Mca-YVADAPK(Dnp)-OHCaspase-1/interleukin-converting enzyme (ICE) and angiotensin I-converting enzyme-2 (ACE-2) substrate3223811 mg13557

 

Coumarin-Labeled Conjugates


Coumarin-labeled conjugates have a wide range of applications in bioimaging and biolabelling due to their bright blue fluoresence and excellent photostability. Coumarin derivatives can be labeled to antibodies, streptavidin, phalloidin, annexin v and other biomoleculces to study apoptosis, label organelles or improve assay sensitivity by amplifying weak signals.
 

Table 7. Available Coumarin-Labeled Conjugates.

Product
Biological Function
Ex (nm)
Em (nm)
Unit Size
Cat No.
iFluor® 350 goat anti-mouse IgG (H+L)Indirectly detects mouse primary antibodies3454501 mg16730
iFluor® 350 goat anti-mouse IgG (H+L) *Cross Adsorbed*Indirectly detects mouse primary antibodies3454501 mg16770
iFluor® 350 goat anti-rabbit IgG (H+L)Indirectly detects rabbit primary antibodies3454501 mg16795
iFluor® 350 goat anti-rabbit IgG (H+L) *Cross Adsorbed*Indirectly detects rabbit primary antibodies3454501 mg16825
iFluor® 350 Goat Anti-human IgG (H+L) AntibodyIndirectly detects human primary antibodies3454501 mg50041
iFluor® 350 Goat Anti-human IgG (H+L) Antibody *Cross Adsorbed*Indirectly detects human primary antibodies3454501 mg50043
iFluor® 350 streptavidin conjugateIndirectly detects biotinylated primary antibodies3454501 mg16980
Annexin V-iFluor® 350 conjugateDetects phosphatidylserine on the cell surface345450100 Tests20070
Phalloidin-iFluor® 350 ConjugateLabels actin filaments (F-actin)345450300 Tests23110
AF350-labeled goat anti-rabbit IgG (H+L)Indirectly detects rabbit primary antibodies3434411 mg16395
AF350 PhalloidinLabels actin filaments (F-actin)343441300 Tests23150
AF350-streptavidin conjugateIndirectly detects biotinylated primary antibodies3434411 mg16890
iFluor® 405 goat anti-rabbit IgG (H+L)Indirectly detects rabbit primary antibodies4034271 mg16796
iFluor® 405 goat anti-rabbit IgG (H+L) *Cross Adsorbed*Indirectly detects rabbit primary antibodies4034271 mg16826
iFluor® 405 goat anti-mouse IgG (H+L)Indirectly detects mouse primary antibodies4034271 mg16731
iFluor® 405 goat anti-mouse IgG (H+L) *Cross Adsorbed*Indirectly detects mouse primary antibodies4034271 mg16771
iFluor® 405 Goat Anti-human IgG (H+L) AntibodyIndirectly detects human primary antibodies4034271 mg50045
iFluor® 405 Goat Anti-human IgG (H+L) *Cross Adsorbed*Indirectly detects human primary antibodies4034271 mg50047
iFluor® 405-streptavidin conjugateIndirectly detects biotinylated primary antibodies4034271 mg16982
Phalloidin-iFluor® 405 ConjugateLabels actin filaments (F-actin)403427300 Tests23111
Annexin V, PacBlue conjugateDetects phosphatidylserine on the cell surface403427100 Tests20089
Phalloidin-AMCA ConjugateLabels actin filaments (F-actin)403427300 Tests23100
DEAC-dUTP *1 mM in Tris Buffer (pH 7.5)*produce dye-containing DNA by conventional enzymatic incorporation methods41147225 nmoles17024