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Fluorogenic Enzyme Substrates Portfolio

Fluorogenic Enzyme Substrates Portfolio

-Enzyme substrates for high-sensitivity fluorescence detection

Fluorogenic enzyme substrates are compounds that generate fluorescence signals upon enzymatic cleavage, enabling high-sensitivity detection of enzyme activity and biological processes. Through enzyme-catalyzed reactions, non-fluorescent or weakly fluorescent substrates are converted into strongly fluorescent products, allowing real-time monitoring and precise analysis.

As a professional supplier of photochemical products, Alfa Chemistry offers a wide range of fluorogenic enzyme substrates, including AMC, MU, CU, Fluorescein, Resorufin, Rhodamine, TFMU, and other custom derivatives. Our products feature diverse specifications, high purity, and excellent batch-to-batch consistency, meeting various research needs and experimental design requirements.

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What Are the Key Features and Benefits?

High Sensitivity

Generates strong fluorescence signals upon enzymatic cleavage, enabling detection of low levels of enzyme activity.

Broad Applicability

Supports the detection of various enzyme types, including proteases, glycosidases, and phosphatases.

Multiple Excitation/Emission Options

Covers blue, green, and red fluorescence channels to meet diverse experimental requirements.

Rapid Reaction Kinetics

Suitable for real-time monitoring and high-throughput screening platforms.

High Purity and Batch Consistency

Ensures reliable and reproducible experimental results.

What Products Do We Offer?

Explore our diverse range of fluorogenic enzyme substrates, each designed for high sensitivity and specific applications in enzymatic activity assays and cellular imaging.

AMC Substrates

CU Substrates

Fluorescein Substrates

MU Substrates

Resorufin Substrates

Rhodamine Substrates

TFMU Substrates

Other Fluorogenic Substrates

What Are the Typical Applications?

To better understand the versatility of these tools, here are some of the most common applications in research and clinical settings:

Enzyme Activity Quantification

Real-time monitoring of enzymatic catalytic efficiency.

Drug and Inhibitor Screening

Rapid screening of potential compounds on high-throughput platforms.

Cell Imaging and Signal Monitoring

Studying intracellular enzyme activity and signaling pathways.

High-Throughput Screening (HTS)

Suitable for drug discovery laboratories.

Clinical Biomarker Analysis

Detection of disease-related enzyme activities.

How Do These Products Compare?

Substrate Type	Excitation/Emission (nm)	Main Applications	Advantages
AMC	360/440	Various enzyme activity assays	Classic blue fluorescence
Fluorescein	~490/515	Cell-based / in vitro assays	High quantum yield
Rhodamine	~540/580	Biological samples	Low background, clear signal
Resorufin	Red	Various enzymes	High signal-to-noise ratio
MU	360/450	Fluorogenic enzyme assays	High sensitivity
TFMU	360/450	High-contrast assays	Enhanced signal contrast

Customers Often Look For

To support diverse research directions and technical requirements, Alfa Chemistry has selected a range of hot-selling fluorogenic enzyme substrates to help researchers efficiently perform enzyme activity assays, drug screening, and cell imaging experiments.

L-Proline 7-amido-4-methylcoumarin hydrobromide

3-Carboxyumbelliferyl β-D-glucuronide

Colchicine fluorescein

4-Methylumbelliferyl α-D-arabinopyranoside

Resorufin acetate

Rhodamine 110 bis-(L-aspartic acid amide) trifluoroacetic acid salt

4-Trifluoromethylumbelliferyl β-D-glucopyranoside

3-Acetylumbelliferyl β-D-glucopyranoside

L-Proline 7-amido-4-methylcoumarin hydrobromide | CAS 115388-93-7

Product Description: Standard L-Proline-AMC substrate with excellent fluorescence performance, a classic choice for protease and peptidase activity studies.
Recommended Applications: Protease and peptidase activity assays, in vitro enzyme kinetics studies.
Packaging: 1 mg, 5 mg, 10 mg

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3-Carboxyumbelliferyl β-D-glucuronide | CAS 216672-17-2

Product Description: High-purity 3-CU substrate with strong fluorescence, commonly used for β-glucuronidase activity detection.
Recommended Applications: Enzyme kinetics studies, β-glucuronidase detection in environmental and biological samples.
Packaging: 1 mg, 5 mg, 10 mg

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Colchicine fluorescein | CAS 66091-34-7

Product Description: Fluorescently labeled colchicine with bright green fluorescence, suitable for cell imaging and microtubule studies.
Recommended Applications: Microtubule dynamics observation, cytoskeleton research, live cell imaging.
Packaging: 0.5 mg, 1 mg, 5 mg

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4-Methylumbelliferyl α-D-arabinopyranoside | CAS 119982-23-9

Product Description: Highly sensitive 4-MU substrate for α-arabinopyranosidase activity detection.
Recommended Applications: Enzymatic assays, glycosidase kinetics studies, metabolic research.
Packaging: 1 mg, 5 mg, 10 mg

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Resorufin acetate | CAS 1152-14-3

Product Description: Standard Resorufin substrate with bright red fluorescence, widely used for hydrolase activity detection.
Recommended Applications: Esterase activity analysis, cell function studies, fluorescent labeling experiments.
Packaging: 1 mg, 5 mg, 10 mg

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Rhodamine 110 bis-(L-aspartic acid amide) trifluoroacetic acid salt | CAS 220846-63-9

Product Description: Rhodamine 110 derivative with bright red fluorescence, suitable for protease and peptidase activity detection.
Recommended Applications: Protease activity analysis, intracellular enzyme studies, fluorescence imaging.
Packaging: 1 mg, 5 mg, 10 mg

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4-Trifluoromethylumbelliferyl β-D-glucopyranoside | CAS 116981-86-3

Product Description: High-purity 4-CF-MU substrate with high fluorescence sensitivity, commonly used for β-glucosidase analysis.
Recommended Applications: Enzyme kinetics studies, β-glucosidase detection in environmental and biological samples.
Packaging: 1 mg, 5 mg, 10 mg

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3-Acetylumbelliferyl β-D-glucopyranoside | CAS 20943-16-2

Product Description: 3-Acetyl-MU substrate with excellent fluorescence performance, suitable for β-glucosidase and esterase detection.
Recommended Applications: Enzyme activity analysis, substrate specificity studies, in vitro experiments.
Packaging: 1 mg, 5 mg, 10 mg

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How Can You Get Technical Support?

Assay Guides / Protocols:

Detailed experimental procedures and practical considerations

Fluorescence Spectral Data:

Reference excitation and emission spectra for each substrate

Let's Talk

What Success Stories Can We Share?

Discover how our products are applied in real-world scenarios through our case studies.

Case 1: Ac-Ile-Glu-Pro-Asp-AMC — Caspase-9 Activity Detection

Background

Caspase-9 is a key upstream enzyme in the apoptotic signaling pathway, and accurate measurement of its activity is critical for drug screening and mechanistic studies. The research team aimed to establish a highly sensitive and quantitative method for Caspase-9 activity detection.

Experimental Design

Ac-Ile-Glu-Pro-Asp-AMC was used as a fluorogenic substrate with high specificity for Caspase-9.
Cell lysates or purified Caspase-9 enzyme solutions were incubated with the substrate at 37 °C for 30 minutes.
Fluorescence signals were measured at excitation/emission wavelengths of 360/440 nm, and fluorescence intensity was recorded over time.

Key Results

Caspase-9 activity was detectable at substrate concentrations as low as 0.5 μM.
Fluorescence intensity showed a strong linear correlation with enzyme concentration (R² = 0.997), enabling reliable quantitative analysis.
The assay successfully differentiated the inhibitory effects of various compounds and validated several known Caspase-9 inhibitors.

Conclusion

Ac-Ile-Glu-Pro-Asp-AMC provides a highly sensitive and specific assay for Caspase-9 activity detection and is well suited for drug screening and apoptosis mechanism studies.

Case 2: Fluorescein di-β-D-glucopyranoside — Imaging of β-Glucosidase Activity

Background

β-Glucosidase plays an important role in plant tissues and microbial metabolism. The research team sought to achieve real-time in situ imaging to investigate the spatial distribution of enzyme activity.

Experimental Design

Fluorescein di-β-D-glucopyranoside was used as a substrate, which releases fluorescein and generates green fluorescence upon enzymatic cleavage by β-glucosidase.
The substrate was added to plant tissue sections or microbial culture systems.
Fluorescence signals were monitored in real time using fluorescence microscopy at excitation/emission wavelengths of approximately 490/515 nm.

Key Results

Clear green fluorescence signals were observed at substrate concentrations as low as 1 μM, with signal intensity increasing proportionally to enzyme activity.
Successful spatial imaging of β-glucosidase activity was achieved across different tissues and cell populations.
The method was non-destructive and suitable for long-term dynamic observation.

Conclusion

Fluorescein di-β-D-glucopyranoside is an ideal tool for real-time imaging of β-glucosidase activity, supporting applications in plant biology and microbiological research.

Case 3: Resorufin β-D-cellobioside — High-Throughput Screening of β-Glucosidase Inhibitors

Background

During drug development, the research team aimed to rapidly screen compounds for their inhibitory effects on β-glucosidase, requiring a high-throughput and sensitive assay method.

Experimental Design

Resorufin β-D-cellobioside was employed as a fluorogenic substrate in a 384-well microplate format for high-throughput screening.
Test compounds and substrate were added to each well and incubated at 37 °C for 20 minutes.
Fluorescence signals were detected in the red channel with emission at approximately 580 nm.

Key Results

Stable fluorescence signals were generated at substrate concentrations as low as 2 μM, showing a linear correlation with enzyme activity (Z'-factor = 0.76).
Hundreds of samples were screened within 2 hours, demonstrating excellent throughput and assay robustness.
Multiple potential inhibitors were successfully identified, providing reliable data for subsequent drug development.

Conclusion

Resorufin β-D-cellobioside is well suited for high-throughput screening of β-glucosidase activity, offering strong fluorescence signals, low background interference, and high experimental efficiency.

Frequently Asked Questions (FAQs)

Q1: What types of enzymes are fluorogenic substrates suitable for?

Fluorogenic enzyme substrates can be used to detect a wide range of enzymes, including proteases, peptidases, glycosidases, phosphatases, and other hydrolases. Different substrate types (such as AMC, MU, Fluorescein, and Rhodamine) are designed for specific enzymes and detection channels, allowing users to select the most appropriate substrate based on experimental requirements.

Q2: What are the recommended storage conditions for these substrates?

Most fluorogenic enzyme substrates should be stored under dry, light-protected, and low-temperature conditions (typically at −20 °C or 4 °C). Repeated freeze–thaw cycles should be avoided to maintain substrate stability and fluorescence performance.

Q3: How do I choose the right fluorogenic substrate for my experiment?

When selecting a fluorogenic substrate, factors such as enzyme type, required detection sensitivity, excitation/emission wavelengths, experimental system (in vitro, cellular, or tissue imaging), and background interference should be considered. Alfa Chemistry offers a broad selection of substrates with different specifications and fluorescence channels to meet diverse research needs.

Q4: What factors influence fluorescence signal intensity?

Fluorescence signal intensity is affected by substrate concentration, enzyme activity, reaction time, pH, and temperature. To achieve optimal results, it is recommended to optimize reaction parameters according to the substrate datasheet and experimental conditions.

Q5: Are fluorogenic enzyme substrates suitable for high-throughput screening (HTS)?

Yes. Most fluorogenic enzyme substrates are designed to be compatible with high-throughput screening platforms and microplate formats (96-, 384-, and 1536-well plates), enabling rapid and sensitive enzyme activity and inhibitor screening.

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