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
for preparing blue fluorescent peptides, proteins and other biomolecules. Compared to fluoresceins
, 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.
Excitation and emission spectra of AMCA, a commonly used coumarin derivative.
Table 1. Spectral properties of 7-Amino-4-trifluoromethylcoumarin.
|Laser Line:||405 nm laser|
|Extinction Coefficient (ε):||17,000 cm-1M-1|
|Quantum Yield (Φ):||0.53|
iFluor® 350 - a superior alternative to AMCA and Alexa Fluor® 350
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. Learn More
Table 2. iFluor® 350 - a superior alternative to AMCA and Alexa Fluor® 350
We offer a broad selection of coumarin products including reactive dyes
for labeling peptides, proteins and other biomolecules, conjugates
for detecting enzymatic activity in cells, homogenates and solution. Learn More
Table 3. Coumarin Reference Standards
Table 4. Coumarin Precursors
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.
|AMCA acid|| || ||346||434||19000||25 mg||501|
|AMCA, succinimidyl ester||Amine||Succinimidyl Ester||346||434||19000||10 mg||502|
|AMCA azide||Alkyne||Azide||346||434||19000||1 mg||508|
|AMCA alkyne||Azide||Alkyne||346||434||19000||1 mg||507|
|AMCA C2 maleimide||Thiol||Maleimide||346||434||19000||5 mg||503|
|AMCA ethylenediamine||Aldehyde, Carboxylic Acid, Ketone||Ethylenediamine||346||434||19000||5 mg||504|
|DEAC||Amine (after pre-activation with carbodiimides)||Carboxylic Acid||411||472||40000||25 mg||505|
|DEAC, succinimidyl ester||Amine||Succinimidyl Ester||411||472||40000||25 mg||506|
|7-Hydroxy-4-methylcoumarin-3-acetic acid|| || ||364||458||25000||100 mg||554|
|7-Hydroxy-4-methylcoumarin-3-acetic acid, succinimidyl ester||Amine||Succinimidyl Ester||364||458||25000||25 mg||556|
- 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.
|Ac-DEVD-AFC *CAS 201608-14-2*||Caspase 3/7 Substrate||376||482||5 mg||13401|
|Ac-DEVD-AMC *CAS 169332-61-0*||Caspase 3/7 Substrate||341||441||5 mg||13402|
|Z-DEVD-AFC||Caspase 3/7 Substrate||376||482||5 mg||13420|
|Z-DEVD-AMC||Caspase 3/7 Substrate||341||441||5 mg||13421|
|Ac-IETD-AFC *CAS 211990-57-7*||Caspase 8 Substrate||376||482||5 mg||13410|
|Ac-IETD-AMC||Caspase 8 Substrate||341||441||5 mg||13411|
|Z-IETD-AFC *CAS 219138-02-0*||Caspase 8 Substrate||376||482||5 mg||13425|
|Ac-LEHD-AMC *CAS 292633-16-0*||Caspase 9 Substrate||341||441||5 mg||13426|
|Gly-Pro-AMC *CAS 115035-46-6*||Dipeptidyl Peptidase IV (DPPIV) Substrate||341||441||5 mg||13450|
|Ala-AMC||Alanyl Aminopeptidase and Trypsin Substrate||341||441||5 mg||13458|
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.
|iFluor® 350 goat anti-mouse IgG (H+L)||Indirectly detects mouse primary antibodies||345||450||1 mg||16730|
|iFluor® 350 goat anti-mouse IgG (H+L) *Cross Adsorbed*||Indirectly detects mouse primary antibodies||345||450||1 mg||16770|
|iFluor® 350 goat anti-rabbit IgG (H+L)||Indirectly detects rabbit primary antibodies||345||450||1 mg||16795|
|iFluor® 350 goat anti-rabbit IgG (H+L) *Cross Adsorbed*||Indirectly detects rabbit primary antibodies||345||450||1 mg||16825|
|iFluor® 350 Goat Anti-human IgG (H+L) Antibody||Indirectly detects human primary antibodies||345||450||1 mg||50041|
|iFluor® 350 Goat Anti-human IgG (H+L) Antibody *Cross Adsorbed*||Indirectly detects human primary antibodies||345||450||1 mg||50043|
|iFluor® 350 streptavidin conjugate||Indirectly detects biotinylated primary antibodies||345||450||1 mg||16980|
|Annexin V-iFluor® 350 conjugate||Detects phosphatidylserine on the cell surface||345||450||100 Tests||20070|
|Phalloidin-iFluor® 350 Conjugate||Labels actin filaments (F-actin)||345||450||300 Tests||23110|
|AF350-labeled goat anti-rabbit IgG (H+L)||Indirectly detects rabbit primary antibodies||343||441||1 mg||16395|