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Amplite® Fluorimetric Glucose Oxidase Assay Kit *Red Fluorescence*

Glucose oxidase dose response was measured with Amplite® Fluorimetric Glucose Oxidase Assay Kit in a solid black 96-well plate using a Gemini fluorescence microplate reader (Molecular Devices).
Glucose oxidase dose response was measured with Amplite® Fluorimetric Glucose Oxidase Assay Kit in a solid black 96-well plate using a Gemini fluorescence microplate reader (Molecular Devices).
Glucose oxidase dose response was measured with Amplite® Fluorimetric Glucose Oxidase Assay Kit in a solid black 96-well plate using a Gemini fluorescence microplate reader (Molecular Devices).
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Telephone1-800-990-8053
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Spectral properties
Excitation (nm)571
Emission (nm)584
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12171501

OverviewpdfSDSpdfProtocol


Excitation (nm)
571
Emission (nm)
584
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 is easily adapted to automation without a separation step. The kit uses our Amplite® Red substrate which enables a dual recordable mode. The fluorescent signal can be easily read by either a fluorescence microplate reader or an absorbance microplate reader. With the Amplite® Fluorimetric Glucose Oxidase Assay Kit, we have detected as little as 0.05 mU/mL glucose oxidase in a 100 µL reaction volume.

Platform


Fluorescence microplate reader

Excitation540 nm
Emission590 nm
Cutoff570 nm
Recommended plateSolid black

Components


Example protocol


AT A GLANCE

Protocol Summary
  1. Prepare glucose oxidase standards or test samples (50 µL)
  2. Add GO working solution (50 µL)
  3. Incubate at 37 °C for 10 - 30 minutes
  4. Monitor fluorescence intensity at Ex/Em = 540/590 nm 
Important      Thaw all the kit components to 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.

1. Amplite™ Red stock solution (250X)
Add 100 µL of DMSO (Component E) into the vial of Amplite™ Red (Component A). The stock solution should be used promptly.
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 standard solution (100 U/mL)
Add 1 mL of Assay Buffer into the vial of Glucose Oxidase (Component D).

4. Glucose stock solution (10X)
Add 5 mL of Assay Buffer 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/11300


Glucose Oxidase standard
Prepare a glucose oxidase standard by diluting 2 µL of the 100 U/mL glucose oxidase standard solution into 200 µL of Assay Buffer (Component B) to have 1000 mU/mL glucose oxidase standard solution. And then take 10 µL of 1000 mU/mL glucose oxidase standard solution and perform 1:100 dilution to obtain 10 mU/mL glucose oxidase standard solution (GOS7). Then perform 1:3 serial dilutions to get remaining serially diluted glucose oxidase standards (GOS6-GOS1). A non-glucose oxidase buffer is included as blank control. The final glucose oxidase concentrations should be twofold lower (i.e., 0 to 5 mU/mL). Note: High concentrations of glucose oxidase may cause reduced fluorescence signal due to the overoxidation of Amplite™ Red (to a non-fluorescent product).

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 a total volume of 5 mL Glucose Oxidase (GO) working solution. Protect from light.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of glucose oxidase standards and test samples in a solid black 96-well microplate. GOS = Glucose Oxidase Standard (GOS1-GOS7, 0.01 to 10 mU/mL), BL = Blank Control, TS = Test Samples.
BLBLTSTS
GOS1GOS1......
GOS2GOS2......
GOS3GOS3
GOS4GOS4
GOS5GOS5
GOS6GOS6
GOS7GOS7
Table 2. Reagent composition for each well.
WellVolumeReagent
GOS1-GOS750 µLserial diltuion (0.01 to 10 mU/mL)
BL50 µLAssay Buffer (Component B)
TS50 µLSample
  1. Prepare glucose oxidase standards (GOS), blank control (BL), and test samples (TS) according ot the layout proided in Table 1 and Table 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.
  2. Add 50 µL of GO working solution into each well of glucose oxidase standards, blank control, and test samples to make the total glucose oxidase 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.
  3. Incubate the reaction for 10 to 30 minutes at 37°C, protected from light.
  4. Monitor the fluorescence intensity with a fluorescence plate reader at Excitation = 530 - 570 nm, Emission = 590 - 600 nm (optimal Ex/Em = 540/590 nm). Note: The contents of the plate can also be transferred to a white clear bottom plate and read by an absorbance microplate reader at the wavelength of 576 ± 5 nm. However, the absorption detection will have a lower sensitivity compared to the fluorescence reading. 

Spectrum


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spectrum

Spectral properties

Excitation (nm)571
Emission (nm)584

Images


Citations


View all 3 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
A reassessment of the carnivorous status of salmonids: hepatic glucokinase is expressed in wild fish in Kerguelen Islands
Authors: Marandel, Lucie and Gaudin, Philippe and Gu{\'e}raud, Fran{\c{c}}ois and Glise, St{\'e}phane and Herman, Alexandre and Plagnes-Juan, Elisabeth and V{\'e}ron, Vincent and Panserat, St{\'e}phane and Labonne, Jacques
Journal: Science of the Total Environment (2018): 276--285

References


View all 29 references: Citation Explorer
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Journal: Photochem Photobiol Sci (2009): 362
Using an indicator displacement assay to monitor glucose oxidase activity in blood serum
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Journal: Org Lett (2007): 1627
Glucose oxidase assisted homogeneous electrochemical receptor binding assay for drug screening
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1,4-Benzoquinone-based electrophoretic assay for glucose oxidase
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Journal: Anal Biochem (2006): 35
Assay for glucose oxidase from Aspergillus niger and Penicillium amagasakiense by Fourier transform infrared spectroscopy
Authors: Karmali K, Karmali A, Teixeira A, Curto MJ.
Journal: Anal Biochem (2004): 320
Comparison of the glucose oxidase method for glucose determination by manual assay and automated analyzer
Authors: Yuen VG, McNeill JH.
Journal: J Pharmacol Toxicol Methods (2000): 543
Interference by gliclazide in the glucose oxidase/peroxidase method for glucose assay
Authors: Nakashima E, Nakamura J, Hamada Y, Koh N, Sakakibara F, Hotta N.
Journal: Diabetes Res Clin Pract (1995): 149
Spectrophotometric assay for superoxide dismutase based on the nitroblue tetrazolium reduction by glucose-glucose oxidase
Authors: Nagi MN, al-Bekairi AM, al-Sawaf HA.
Journal: Biochem Mol Biol Int (1995): 633
Cytophotometric assay of cytochrome oxidase, lactate dehydrogenase and glucose-6-phosphate dehydrogenase activities in human peroxidized spermatozoa
Authors: Ferr, undefined and i B, Cremonesi F, Consiglio AL, Carnevali A, Porcelli F.
Journal: Acta Histochem (1992): 363