Amplite® Fluorimetric Glutamate Oxidase Assay Kit *Red Fluorescence*
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Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
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International | See distributors |
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Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
Excitation (nm) | 571 |
Emission (nm) | 584 |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12171501 |
Overview | SDSProtocol |
Excitation (nm) 571 | Emission (nm) 584 |
Platform
Fluorescence microplate reader
Excitation | 540 nm |
Emission | 590 nm |
Cutoff | 570 nm |
Recommended plate | Solid black |
Components
Example protocol
AT A GLANCE
Protocol summary
- Glutamate Oxidase standards or test samples (50 µL)
- Add Glutamate Oxidase working solution (50 µL)
- Incubate at room temperature for 30 - 60 min
- Read fluorescence intensity at Ex/Em = 540/590 nm
Important notes
Thaw all the kit components to room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
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.
PREPARATION OF STANDARD SOLUTION
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).
PREPARATION OF WORKING SOLUTION
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.
SAMPLE EXPERIMENTAL PROTOCOL
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.
BL | BL | TS | TS |
GO1 | GO1 | ... | ... |
GO2 | GO2 | ... | ... |
GO3 | GO3 | ||
GO4 | GO4 | ||
GO5 | GO5 | ||
GO6 | GO6 | ||
GO7 | GO7 |
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).
Well | Volume | Reagent |
GO1 - GO7 | 50 µL | Serial Dilution (0.01 to 10 mU/mL) |
BL | 50 µL | Assay Buffer (Component B) |
TS | 50 µL | test sample |
- 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.
- 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.
- Incubate the reaction for 30 to 60 minutes at room temperature, protected from light.
- 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.
Product Family
Images
Citations
Authors: Li, Feng and Kuang, Yangduo and Liu, Na and Ge, Fei
Journal: Science of The Total Environment (2019)
References
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Journal: Indian J Exp Biol (2006): 392
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Journal: Anal Chem (2006): 2456
Application notes
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