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Amplite® Fluorimetric Amyloglucosidase Assay Kit *Red Fluorescence*
Product key features
The Amplite® Fluorimetric Amyloglucosidase Assay Kit offers a fast, convenient, and sensitive method for measuring amyloglucosidase activity in biological samples.
- Simple and direct detection: Employs an enzymatic reaction followed by a fluorescence-based detection to specifically quantify amyloglucosidase activity.
- Red fluorescence readout: Generates a bright and stable red fluorescent signal, detectable with standard fluorescence microplate readers.
- Broad sample compatibility: Suitable for various sample types, including enzyme preparations and biological extracts.
Product description
The Amplite® Fluorimetric Amyloglucosidase Assay Kit provides a streamlined and sensitive assay for detecting amyloglucosidase activity through a fluorescence-based approach. In this assay, amyloglucosidase catalyzes a reaction that produces a detectable product, which then undergoes a secondary reaction to generate a red fluorescent signal. The fluorescence intensity is directly proportional to the enzyme activity and can be easily measured using a standard fluorescence microplate reader with appropriate excitation and emission settings.
This kit is compatible with a wide range of enzyme sources and biological samples, making it ideal for enzyme characterization, inhibitor screening, and metabolic research. The assay protocol is simple to perform and yields a stable fluorescent signal, allowing for amyloglucosidase detection in high-throughput screening or routine amyloglucosidase activity analysis.
Example protocol
AT A GLANCE
- Prepare test samples along with serially diluted Amyloglucosidase standards (10 μL).
- Add Amyloglucosidase Substrate working solution (40 μL).
- Incubate at RT for 30 minutes.
- Add Amyloglucosidase Detection working solution (50 μL).
- Incubate at RT for 30 minutes.
- Measure the fluorescence intensity at Ex/Em = 540/590 nm.
Note: Thaw all the kit components at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTIONS
Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles
Add 100 µL of DMSO (Component G) into Amplite® Red (Component A) to make 100X Amplite™ Red stock solution
Add 100 µL distilled water to Amyloglucosidase Detection Enzyme (Component E) to make 50X Amyloglucosidase Detection Enzyme Stock Solution.
Add 100 µL distilled water to Amyloglucosidase Standard (Component F) to make a 10U/mL stock solution.
PREPARATION OF STANDARD SOLUTIONS
https://www.aatbio.com/tools/serial-dilution/13817
PREPARATION OF WORKING SOLUTION
Add 20 µL Amyloglucosidase Substrate (Component C) to 800 µL of Amyloglucosidase Reaction Buffer (Component B) to make Amyloglucosidase Substrate working solution.
Note: 800 µL Amyloglucosidase Substrate working solution is for 20 assays with 96-well plates. Please prepare the volume as needed proportionally. The working solution is not stable, prepare it freshly and use promptly.
Add 10 µL of 100X Amplite® Red stock solution, 20 µL of 50X Amyloglucosidase Detection Enzyme stock solution in 970 µL of Assay Buffer (Component D).
Note: 1mL Amyloglucosidase Detection Enzyme Stock Solution is for 20 assays with 96-well plates. Please prepare the volume as needed proportionally. The working solution is not stable, use it promptly and avoid direct exposure to light.
SAMPLE EXPERIMENTAL PROTOCOL
- Prepare Amyloglucosidase standards (STD1-7), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 96-well plate, add 10 µL per well, and for a 384-well plate, use 5 µL per well instead.
- Add 40 µL of Amyloglucosidase Substrate working solution to each well of blank control, positive control and test samples. For a 384-well plate, add 20 µL working solution into each well instead.
Note: The final Amyloglucosidase concentration in each well are 30, 15, 7.5, 3.75, 1.88, 0.94, 0.468 mU/mL after adding Amyloglucosidase Substrate working solution. - Incubate at RT for 30 minutes.
- Add 50 µL of Amyloglucosidase Detection working solution to each well of blank control, positive control and test samples. For a 384-well plate, add 25 µL working solution into each well instead.
- Incubate at RT for 30 minutes.
- Measure fluorescence intensity at Ex/Em = 540/590 nm (cutoff=570nm).
Table 1. Layout of Amyloglucosidase standards and test samples in a clear bottom 96-well microplate. STD = Amyloglucosidase Standards (STD1-STD7, 2.34 to 150 mU/mL), BL= Blank Control, TS = Test Samples.
BL | BL | Positive Control | Cell content |
STD 1 | STD 1 | ... | ... |
STD 2 | STD 2 | ... | ... |
STD 3 | STD 3 | ||
STD 4 | STD 4 | ||
STD 5 | STD 5 | ||
STD 6 | STD 6 | ||
STD 7 | STD 7 |
Table 2. Reagent composition for each well.
Well | Volume | Reagent |
STD1-STD7 | 40 µL | Serial Dilutions (2.34 to 150mU/mL) |
BL | 40 µL | Amyloglucosidase Reaction Buffer |
TS | 40 µL | Test Sample |
Spectrum
References
Authors: Zhang, Shiyi and de Jonge, Leon and de Vries, Sonja and Gerrits, Walter J J
Journal: Journal of animal science (2025)
Authors: Pasin, Thiago Machado and Betini, Jorge Henrique Almeida and de Lucas, Rosymar Coutinho and Polizeli, Maria de Lourdes Teixeira de Moraes
Journal: Biotechnology progress (2024): e3384
Authors: Yao, Muzi and Liu, Jiahui and Liu, Jiaming and Qi, Xinmiao and Bai, Erlu and Yin, Jinjin and Wu, Tao
Journal: International journal of biological macromolecules (2024): 134974
Authors: Zhang, Kai and Tian, Xiaojing and Shen, Ruixi and Wang, Yang and Zhang, Yafei and Wang, Wenhang
Journal: Food research international (Ottawa, Ont.) (2023): 113053
Authors: Wu, Kaijuan and Zhai, Xingyu and Chen, Hao and Zheng, Jinfeng and Yu, Zheng and Xu, Xuewei and Huang, Jing
Journal: Scientific reports (2023): 5840
Safety data sheet