Amplite® Colorimetric Glucose Oxidase Assay Kit

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Telephone	1-800-990-8053
Fax	1-800-609-2943
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Spectral properties

Excitation (nm)	571
Emission (nm)	584

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
UNSPSC	12171501

Overview SDS Protocol

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 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.

Platform

Absorbance microplate reader

Absorbance	570 nm
Recommended plate	Clear bottom

Components

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

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).
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

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.

BL	BL	TS	TS
GOS1	GOS1	...	...
GOS2	GOS2	...	...
GOS3	GOS3
GOS4	GOS4
GOS5	GOS5
GOS6	GOS6
GOS7	GOS7

Table 2. Reagent composition for each well.

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

Open in Advanced Spectrum Viewer

Spectral properties

Excitation (nm)	571
Emission (nm)	584

Product Family

Name	Excitation (nm)	Emission (nm)
Amplite® Fluorimetric Glucose Oxidase Assay Kit Red Fluorescence	571	584
Amplite® Colorimetric Xanthine Oxidase Assay Kit	571	584

Images

Figure 1. Glucose oxidase dose response was measured with Amplite® Colorimetric Glucose Oxidase Assay Kit (Cat#11299) on a 96-well clear bottom plate using a SpectraMax reader (Molecular Devices) with path check on.

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, Fran&ccaron;ois 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

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

Conformation and activity dependent interaction of glucose oxidase with CdTe quantum dots: towards developing a nanoparticle based enzymatic assay
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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

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

1,4-Benzoquinone-based electrophoretic assay for glucose oxidase
Authors: Urban PL, Goodall DM, Bergstrom ET, Bruce NC.
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