Amplite® Colorimetric Glucose Quantitation Kit

Glucose, a monosaccharide, is the most important carbohydrate in biology. It is a source of energy and metabolic intermediate for cell growth. As one of the main products of photosynthesis, glucose starts cellular respiration in both prokaryotes and eukaryotes. Glucose level is a key diagnostic parameter for many metabolic disorders, e.g., diabetes. This Amplite® Colorimetric Glucose Quantitation Kit provides a quick and sensitive method for the measurement of glucose. It uses glucose oxidase-based enzyme coupled reactions to detect glucose through the production of hydrogen peroxide, which is monitored by our Amplite® Red peroxidase substrate. Amplite® Red peroxidase substrate can be read by an absorbance microplate reader at ~570 nm. The assay is robust, and can be readily adapted for a wide variety of applications that require the measurement of glucose. The assay has very low background since it is run in the red visible range that significantly reduces the interference from biological samples. With the Amplite® Colorimetric Glucose Quantitation Kit, we can detect as little as 3 µM D-glucose.

Example protocol

AT A GLANCE

Protocol Summary

Prepare Glucose standards and/or test samples (50 µL)
Add Glucose Assay working solution (50 µL)
Incubate at 37°C for 10 - 30 minutes
Monitor absorbance increase at OD 570±5 nm

Important 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.

1. Amplite™ Red stock solution (250X)

Add 100 µL of DMSO (Component E) into the vial of Amplite™ Red substrate (Component A). The stock solution should be used promptly. Any remaining solution should be aliquoted and refrozen at -20 ^oC.
Note Avoid repeated freeze-thaw cycles. Note: The Amplite™ Red substrate 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 substrate 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. Horseradish Peroxidase (HRP) stock solution (10 U/mL)

Add 1 mL of Assay Buffer (Component B) into the vial of Horseradish Peroxidase (Component C).
Note The unused HRP solution should be divided into single use aliquotes and stored at -20 ^oC.

3. Glucose Oxidase solution (100 U/mL)

Add 1 mL of Assay Buffer (Component B) into the vial of Glucose Oxidase (Component D).
Note The unused Glucose Oxidase solution should be divided into single use aliquotes and stored at -20 ^oC.

4. Glucose stock solution (800mM)

Add 1 mL of Assay Buffer (Component B) into the vial of Glucose (Component F).
Note The unused Glucose solution should be divided into single use aliquotes and stored at -20 ^oC.

PREPARATION OF STANDARD SOLUTION

For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/40004

Glucose standard

Prepare a glucose standard by diluting the appropriate amount of the 800 mM glucose stock solution into assay buffer (Component B) to produce glucose concentrations of 100 µM. Then perform 1:2 serial dilutions in assay buffer (Component B) to get approximately 50, 25, 12.5, 6.3, 3.1 and 1.6 µM serially diluted glucose standards. A non-glucose buffer control is included as blank control.

PREPARATION OF WORKING SOLUTION

Table 1.Assay working solution for one clear bottom 96-well microplate (2X)

Components	Volume
Amplite&trade Red Stock Solution (250x)	20 µL
HRP Stock Solution (10 U/mL)	100 µL
Glucose Oxidase Solution (100 U/mL)	100 µL
Assay Buffer	4.78 mL
Total volume	5 mL

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of Glucose standards and test samples in a clear bottom 96-well microplate. GS = Glucose standard (GS1-GS7); BL = blank control; TS = test sample.

BL	BL	TS	TS
GS1	GS1	...	...
GS2	GS2	...	...
GS3	GS3
GS4	GS4
GS5	GS5
GS6	GS6
GS7	GS7

Table 2. Reagent composition for each well

Well	Volume	Reagent
GS1 - GS7	50 µL	Serial Dilutions (1.6 -100 µM)
BL	50 µL	Assay Buffer (Compound B)
TS	50 µL	Test Sample

Glucose assay

Add glucose standards and glucose containing test samples into a 96-well clear bottom microplate as described in Tables 2 and 3.
Add 50 µL of Assay working solution into each well of glucose standard, blank control, and test samples (Table 2) to make the total glucose assay volume of 100 µL/well.
Note For a 384-well plate, add 25 µL of sample and 25 µL of assay reaction mixture into each well.
Incubate the reaction for 10 to 30 minutes at 37 ^oC, protected from light.
Monitor the absorbance increase with an absorbance plate reader at OD = 570 nm.

Spectrum

Open in Advanced Spectrum Viewer

Citations

View all 11 citations: Citation Explorer

Female Wistar Kyoto More Immobile rats with genetic stress hyper-reactivity show enhanced contextual fear memory without deficit in extinction of fear
Authors: Harter, Aspen M and Nemesh, Mariya and Ji, Michelle T and Lee, Luca and Yamazaki, Anna and Kim, Chris and Redei, Eva E
Journal: European Journal of Neuroscience (2024)

RIP140 inhibits glycolysis-dependent proliferation of breast cancer cells by regulating GLUT3 expression through transcriptional crosstalk between hypoxia induced factor and p53
Authors: Jacquier, Valentin and Gitenay, Delphine and Fritsch, Samuel and Bonnet, Sandrine and Gy{\H{o}}rffy, Bal{\'a}zs and Jalaguier, St{\'e}phan and Linares, Laetitia K and Cavaill{\`e}s, Vincent and Teyssier, Catherine
Journal: Cellular and Molecular Life Sciences (2022): 1--17

Aspects {\'e}volutifs et environnementaux de la plasticit{\'e} ph{\'e}notypique chez deux Moronid{\'e}s, le bar Europ{\'e}en (Dicentrarchus labrax) et le bar ray{\'e} (Morone saxatilis)
Authors: Gourtay, Clémence
Journal: (2018)

MicroRNAs regulate gene plasticity during cold shock in zebrafish larvae
Authors: Hung, I-Chen and Hsiao, Yu-Chuan and Sun, H Sunny and Chen, Tsung-Ming and Lee, Shyh-Jye
Journal: BMC genomics (2016): 922

Early nutritional programming in fish: tailoring the metabolic use of dietary carbohydrates
Authors: Rocha, Filipa Soares
Journal: (2015)

References

View all 21 references: Citation Explorer

Insulin and glucose mediate opposite intracellular ionized magnesium variations in human lymphocytes
Authors: Delva P, Degan M, Trettene M, Lechi A.
Journal: J Endocrinol (2006): 711

Recombinant human glucose-6-phosphate dehydrogenase. Evidence for a rapid-equilibrium random-order mechanism
Authors: Wang XT, Au SW, Lam VM, Engel PC.
Journal: Eur J Biochem (2002): 3417

Fluorescent microplate cell assay to measure uptake and metabolism of glucose in normal human lung fibroblasts
Authors: Leira F, Louzao MC, Vieites JM, Botana LM, Vieytes MR.
Journal: Toxicol In Vitro (2002): 267

Glucose-induced alterations of intracellular ionized magnesium in human lymphocytes
Authors: Delva P, Degan M, Pastori C, Faccini G, Lechi A.
Journal: Life Sci (2002): 2119

Plasma glycohydrolase levels in patients with type 1 diabetes at onset and in subjects undergoing an intravenous glucose tolerance test
Authors: Goi G, Bairati C, Burlina A, Massaccesi L, Monciotti C, Segalini G, Testa R, Lombardo A.
Journal: Metabolism (2000): 1352

Page updated on November 20, 2024

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