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Amplite® Fluorimetric Glutamate Oxidase Assay Kit *Red Fluorescence*

Glutamate oxidase dose response was measured with Amplite® Fluorimetric Glutamate Oxidase Assay Kit in a 96-well solid black plate using a Gemini fluorescence microplate reader (Molecular Devices).
Glutamate oxidase dose response was measured with Amplite® Fluorimetric Glutamate Oxidase Assay Kit in a 96-well solid black plate using a Gemini fluorescence microplate reader (Molecular Devices).
Glutamate oxidase dose response was measured with Amplite® Fluorimetric Glutamate Oxidase Assay Kit in a 96-well solid black plate using a Gemini fluorescence microplate reader (Molecular Devices).
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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


Excitation (nm)
Emission (nm)
Glutamate oxidase belongs to the family of oxidoreductases, specifically those acting on the CH-NH2 group of donors with oxygen as an acceptor. It is an enzyme that specifically catalyzes the oxidative deamination of L-glutamate in the presence of water and oxygen with the formation of o-ketoglutarate, ammonia, and hydrogen peroxide. The Amplite® Fluorimetric Glutamate Oxidase Assay Kit provides a quick and ultrasensitive method for the measurement of glutamate oxidase in solution and in cell lysates. In the assay, L-glutamic acid is oxidized to µ-ketoglutarate, NH3 and H2O2 by glutamate oxidase. L-Alanine and L-glutamate-pyruvate transaminase are included in the reaction, resulting in multiple cycles of the initial reaction, thus significantly amplifying the production of H2O2. 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® Glutamate Oxidase Assay kit, we have detected as little as 40 uU/mL glutamate oxidase in a 100 µL reaction volume. It can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation without a separation step.


Fluorescence microplate reader

Excitation540 nm
Emission590 nm
Cutoff570 nm
Recommended plateSolid black


Example protocol


Protocol summary

  1. Glutamate Oxidase standards or test samples (50 µL)
  2. Add Glutamate Oxidase working solution (50 µL)
  3. Incubate at room temperature for 30 - 60 min
  4. Read fluorescence intensity at Ex/Em = 540/590 nm

Important notes
Thaw all the kit components to room temperature before starting the experiment.


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 40 µL of DMSO (Component F) 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 (400X):
Add 200 µL of Assay Buffer (Component B) into the vial of Horseradish Peroxidase (Component C).

3. Glutamic Acid stock solution (400X):
Add 1.0 mL of ddH2O into the vial of Glutamic Acid (Component D) to make 400X glutamic acid stock solution.

4. Glutamate Oxidase (GO) standard solution (150 mU/mL):
Add 100 µL of Assay Buffer (Component B) into the vial of Glutamate Oxidase Standard (lyophilized, Component E) to make 150 mU/mL Glutamate Oxidase (GO) standard solution.


Glutamate Oxidase standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/11302

Add 30 µL of 150 mU/mL GO standard solution into 420 µL of Assay Buffer (Component B) to get 10 mU/mL GO standard solution (GO7). Take 10 mU/mL GO standard solution to perform 1:3 serial dilutions to get remaining serially diluted GO standards (GO6 - GO1).


Add 20 μL of Amplite™ Red stock solution (250X), 12.5 μL of HRP stock solution (400X) and 12.5 μL of Glutamic Acid stock solution (400X) into 5 mL of Assay Buffer (Component B) to make a total volume of 5.07 mL Glutamate Oxidase working solution(GO working solution). Protect from light.


Table 1. Layout of GO standards and test samples in a solid black 96-well microplate. GO= glutamate oxidase standards (GO1 - GO7, 0.01 to 10 mU/mL), BL=blank control, TS=test samples.


Table 2. Reagent composition for each well. Higher concentrations of GO may cause reduced fluorescence signal due to the over oxidation of Amplite™ Red (to a non-fluorescent product).

GO1 - GO750 µLSerial Dilution (0.01 to 10 mU/mL)
BL50 µLAssay Buffer (Component B)
TS50 µLtest sample
  1. Prepare GO standards (GO), 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.

  2. Add 50 µL of GO working solution to each well of GO standard, blank control, and test samples to make the total GO 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 30 to 60 minutes at room temperature, 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), cutoff = 570 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. The absorption detection has lower sensitivity compared to fluorescence reading.


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Spectral properties

Excitation (nm)571
Emission (nm)584



View all 1 citations: Citation Explorer
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Authors: Li, Feng and Kuang, Yangduo and Liu, Na and Ge, Fei
Journal: Science of The Total Environment (2019)


View all 65 references: Citation Explorer
Structural characterization of L-glutamate oxidase from Streptomyces sp. X-119-6
Authors: Arima J, Sasaki C, Sakaguchi C, Mizuno H, Tamura T, Kashima A, Kusakabe H, Sugio S, Inagaki K.
Journal: Febs J (2009): 3894
Visualizing L-glutamate fluxes in acute hippocampal slices with glutamate oxidase-immobilized coverslips
Authors: Okumura W, Moridera N, Kanazawa E, Shoji A, Hirano-Iwata A, Sugawara M.
Journal: Anal Biochem (2009): 326
Coupling of energy metabolism and synaptic transmission at the transcriptional level: role of nuclear respiratory factor 1 in regulating both cytochrome c oxidase and NMDA glutamate receptor subunit genes
Authors: Dhar SS, Wong-Riley MT.
Journal: J Neurosci (2009): 483
Activation of metabotropic glutamate receptor 5 modulates microglial reactivity and neurotoxicity by inhibiting NADPH oxidase
Authors: Loane DJ, Stoica BA, Pajoohesh-Ganji A, Byrnes KR, Faden AI.
Journal: J Biol Chem (2009): 15629
Nuclear respiratory factor 1 co-regulates AMPA glutamate receptor subunit 2 and cytochrome c oxidase: tight coupling of glutamatergic transmission and energy metabolism in neurons
Authors: Dhar SS, Liang HL, Wong-Riley MT.
Journal: J Neurochem (2009): 1595
A D-pathway mutation decouples the Paracoccus denitrificans cytochrome c oxidase by altering the side-chain orientation of a distant conserved glutamate
Authors: Durr KL, Koepke J, Hellwig P, Muller H, Angerer H, Peng G, Olkhova E, Richter OM, Ludwig B, Michel H.
Journal: J Mol Biol (2008): 865
Glutamate 107 in subunit I of the cytochrome bd quinol oxidase from Escherichia coli is protonated and near the heme d/heme b595 binuclear center
Authors: Yang K, Zhang J, Vakkasoglu AS, Hielscher R, Osborne JP, Hemp J, Miyoshi H, Hellwig P, Gennis RB.
Journal: Biochemistry (2007): 3270
The efficiency of immobilised glutamate oxidase decreases with surface enzyme loading: an electrostatic effect, and reversal by a polycation significantly enhances biosensor sensitivity
Authors: McMahon CP, Rocchitta G, Serra PA, Kirwan SM, Lowry JP, O'Neill RD.
Journal: Analyst (2006): 68
Development of biosensor based on immobilized L-glutamate oxidase for determination of monosodium glutamate in food
Authors: Basu AK, Chattopadhyay P, Roychudhuri U, Chakraborty R.
Journal: Indian J Exp Biol (2006): 392
Improving the performance of glutamate microsensors by purification of ascorbate oxidase
Authors: Oldenziel WH, de Jong LA, Dijkstra G, Cremers TI, Westerink BH.
Journal: Anal Chem (2006): 2456