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

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

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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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Coumarin Products

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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

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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

1. ε = molar extinction coefficient at their maximum absorption wavelength (Units = cm^-1M^-1).

Coumarin-Labeled Enzyme Substrates

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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.

Coumarin-Labeled Conjugates

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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.