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Amplite® Colorimetric Glucose Oxidase Assay Kit
The glucose oxidase is a dimeric protein that catalyzes the oxidation of beta-D-glucose into hydrogen peroxide and D-glucono-1,5-lactone, which is hydrolyzed to gluconic acid. It is widely used for the determination of glucose in body fluids and in removing residual glucose and oxygen from beverages, food and other agricultural products. Furthermore, Glucose oxidase is commonly used in biosensors to detect glucose. The Amplite® Glucose Oxidase Assay Kit provides a quick and sensitive method for the measurement of glucose oxidase in solution. It can be performed in a convenient 96-well or 384-well microtiter plate format and readily adapted to automation without a separation step. The kit uses our Amplite® Red substrate which can be monitored using an absorbance microplate reader at 570 nm.
Example protocol
AT A GLANCE
Protocol Summary
- Prepare glucose oxidase standards or test samples (50 µL)
- Add working solution (50 µL)
- Incubate at 37 °C for 10 - 30 minutes
- Monitor absorbance at OD = 570 nm
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.
Note The Amplite™ Red is unstable in the presence of thiols such as dithiothreitol (DTT) and 2-mercaptoethanol. The final concentration of DTT or 2-mercaptoethanol in the reaction should be no higher than 10 µM. The Amplite™ Red is also unstable at high pH (>8.5). Therefore, the reaction should be performed at pH 7 – 8. The provided assay buffer (pH 7.4) is recommended.
1. Amplite™ Red stock solution (250X)
Add 100 µL of DMSO (Component E) into the vial of Amplite™ Red (Component A). Note The Amplite™ Red is unstable in the presence of thiols such as dithiothreitol (DTT) and 2-mercaptoethanol. The final concentration of DTT or 2-mercaptoethanol in the reaction should be no higher than 10 µM. The Amplite™ Red is also unstable at high pH (>8.5). Therefore, the reaction should be performed at pH 7 – 8. The provided assay buffer (pH 7.4) is recommended.
2. HRP stock solution (50X)
Add 1 mL of Assay Buffer (Component B) into the vial of Horseradish Peroxidase (Component C).3. Glucose oxidase stock solution (100 U/mL)
Add 1 mL of Assay Buffer (Component B) into the vial of Glucose Oxidase (Component D).4. Glucose stock solution (10X)
Add 5 mL of Assay Buffer (Component B) into the vial of Glucose (Component F).PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/11299
https://www.aatbio.com/tools/serial-dilution/11299
Glucose Oxidase standard
Prepare a glucose oxidase standard by diluting 2 µL of the 100 U/mL Glucose Oxidase stock solution into 200 µL of Assay Buffer (Component B) to have 1000 mU/mL glucose oxidase standard solution. Then perform 1:100 serial dilution followed by 1:2 serial dilutions to get serially diluted glucose oxidase standards from 10 mU/mL to 0.156 mU/mL (GOS1 - GOS7).PREPARATION OF WORKING SOLUTION
Add 20 µL of Amplite™ Red stock solution (250X), 100 µL of HRP stock solution (50X), and 500 µL of Glucose stock solution (10X) into 4.4 mL of Assay Buffer (Component B) to make 5 mL of working solution.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of glucose oxidase standards and test samples in a clear bottom 96-well microplate. GOS = Glucose Oxidase Standards (GOS1 - GOS7, 0.156 to 10 mU/mL), BL = Blank Control, TS = Test Samples.
Table 2. Reagent composition for each well.
| BL | BL | TS | TS |
| GOS1 | GOS1 | ... | ... |
| GOS2 | GOS2 | ... | ... |
| GOS3 | GOS3 | ||
| GOS4 | GOS4 | ||
| GOS5 | GOS5 | ||
| GOS6 | GOS6 | ||
| GOS7 | GOS7 |
| Well | Volume | Reagent |
| GOS1 - GOS7 | 50 µL | Serial Dilution (0.156 to 10 mU/mL) |
| BL | 50 µL | Assay Buffer (Component B) |
| TS | 50 µL | test sample |
- Prepare glucose oxidase standards (GOS), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.
- Add 50 µL of working solution to each well of glucose oxidase standard, blank control, and test samples to make the total assay volume of 100 µL/well. For a 384-well plate, add 25 µL of GO working solution into each well instead, for a total volume of 50 µL/well.
- Incubate the reaction for 10 to 30 minutes at 37 °C, protected from light.
- Monitor the absorbance increase with an absorbance plate reader at OD = 570 nm.
Spectrum
Product family
| Name | Excitation (nm) | Emission (nm) |
| Amplite® Colorimetric Xanthine Oxidase Assay Kit | 571 | 584 |
| Amplite® Fluorimetric Glucose Oxidase Assay Kit *Red Fluorescence* | 571 | 584 |
Citations
View all 2 citations: Citation Explorer
A reassessment of the carnivorous status of salmonids: Hepatic glucokinase is expressed in wild fish in Kerguelen Islands
Authors: Mar, undefined and el, Lucie and Gaudin, Philippe and Guéraud, Frančois and Glise, Stéphane and Herman, Alex and re , undefined and Plagnes-Juan, Elisabeth and Véron, Vincent and Panserat, Stéphane and Labonne, Jacques
Journal: Science of The Total Environment (2018): 276--285
Authors: Mar, undefined and el, Lucie and Gaudin, Philippe and Guéraud, Frančois and Glise, Stéphane and Herman, Alex and re , undefined and Plagnes-Juan, Elisabeth and Véron, Vincent and Panserat, Stéphane and Labonne, Jacques
Journal: Science of The Total Environment (2018): 276--285
Overcoming 5-Fu resistance in human non-small cell lung cancer cells by the combination of 5-Fu and cisplatin through the inhibition of glucose metabolism
Authors: Zhao, Jun-gang and Ren, Kai-ming and Tang, Jun
Journal: Tumor Biology (2014): 12305--12315
Authors: Zhao, Jun-gang and Ren, Kai-ming and Tang, Jun
Journal: Tumor Biology (2014): 12305--12315
References
View all 29 references: Citation Explorer
Factors affecting accuracy and time requirements of a glucose oxidase-peroxidase assay for determination of glucose
Authors: Hall MB, Keuler NS.
Journal: J AOAC Int (2009): 50
Authors: Hall MB, Keuler NS.
Journal: J AOAC Int (2009): 50
Conformation and activity dependent interaction of glucose oxidase with CdTe quantum dots: towards developing a nanoparticle based enzymatic assay
Authors: Priyam A, Chatterjee A, Bhattacharya SC, Saha A.
Journal: Photochem Photobiol Sci (2009): 362
Authors: Priyam A, Chatterjee A, Bhattacharya SC, Saha A.
Journal: Photochem Photobiol Sci (2009): 362
Using an indicator displacement assay to monitor glucose oxidase activity in blood serum
Authors: Zhang T, Anslyn EV.
Journal: Org Lett (2007): 1627
Authors: Zhang T, Anslyn EV.
Journal: Org Lett (2007): 1627
Glucose oxidase assisted homogeneous electrochemical receptor binding assay for drug screening
Authors: Funabashi H, Tanaka Y, Imamura Y, Mie M, Manabe T, Tanaka H, Takahashi T, H and a H, Aizawa M, Kobatake E.
Journal: Biosens Bioelectron (2006): 1675
Authors: Funabashi H, Tanaka Y, Imamura Y, Mie M, Manabe T, Tanaka H, Takahashi T, H and a H, Aizawa M, Kobatake E.
Journal: Biosens Bioelectron (2006): 1675
1,4-Benzoquinone-based electrophoretic assay for glucose oxidase
Authors: Urban PL, Goodall DM, Bergstrom ET, Bruce NC.
Journal: Anal Biochem (2006): 35
Authors: Urban PL, Goodall DM, Bergstrom ET, Bruce NC.
Journal: Anal Biochem (2006): 35
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