Amplite™ Fluorimetric Oxaloacetate Assay Kit *Red Fluorescence*

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Oxaloacetate dose response was measured with the Amplite™ Fluorimetric Oxaloacetate Assay Kit on a solid black 96-well plate using a Gemini microplate reader (Molecular Devices).
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200 Tests 13841 $345


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Overview

Ex/Em (nm)571/585
Storage Freeze (<-15 °C)
Minimize light exposure
InstrumentsFluorescence microplate reader
Category Cell Biology
Cell Metabolism
Related Redox Enzymes
Oxaloacetate is an important part of citric acid cycle, where it reacts with Acetyl-CoA to form citrate. It is also involved in gluconeogenesis, urea cycle, glyoxylate cycle, amino acid synthesis, and fatty acid synthesis. The lack of oxaloacetate limits gluconeogenesis and urea cycle function, and can lead to decreased production of energy. Oxaloacetate can be also used as blood glutamate scavengers to provide neuroprotection after traumatic brain injury, expressed both by reduced neuronal loss in the hippocampus and improved neurologic outcomes. Amplite™ Fluorimetric Oxaloacetate Assay Kit offers a sensitive assay for quantifying oxaloacetate in biological samples. Oxaloacetate is converted to pyruvate that generates hydrogen peroxide through an enzyme coupled reaction. The production of hydrogen peroxide is monitored with Amplite™ Red by a fluorescence microplate reader at Ex/Em = 540/590 nm.




Protocol


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This protocol only provides a guideline, and should be modified according to your specific needs.
At a glance

Protocol summary

  1. Prepare Oxaloacetate standards or test samples (50 µL)
  2. Add Oxaloacetate working solution (50 µL)
  3. Incubate at room temperature for 30 - 60 minutes
  4. Monitor fluorescence increase at Ex/Em = 540/590 nm (Cutoff = 570 nm)

Important notes
Thaw one vial of each kit component at room temperature before starting the experiment.

Key parameters
Instrument:Fluorescence microplate reader
Excitation:540 nm
Emission:590 nm
Cutoff:570 nm
Recommended plate:Solid black
Preparation of stock solution
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. Oxaloacetate standard solution (100 mM):
Add 100 µL of ddH2O into Oxaloacetate Standard (Component E) to make 100 mM Oxaloacetate standard solution.

2. Amplite™ Red substrate stock solution (200X):
Add 50 µL of DMSO (Component F) into Amplite™ Red substrate (Component A) to make 200X Amplite™ Red Substrate stock solution.

3. Oxaloacetate Decarboxcylase (OAC) stock solution (100X):
Add 50 µL of ddH2O into Oxaloacetate Decarboxcylase (Component D) to make 100X Oxaloacetate Decarboxcylase stock solution.

Preparation of standard solution
Oxaloacetate standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/13841

Add 10 μL of 100 mM Oxaloacetate standard solution into 990 μL of Assay Buffer (Component C) to get 1 mM Oxaloacetate standard solution. Take 1 mM Oxaloacetate standard solution and perform 1:10 in Assay Buffer (Component C) to make 100 µM Oxaloacetate standard solution (SD7). Take 100 µM Oxaloacetate standard solution (SD7) and perform 1:3 serial dilutions in Assay Buffer (Component C) to get serially diluted Oxaloacetate standards (SD6 - SD1).

Preparation of working solution

1. Add 5mL Assay Buffer (Component C) into one Enzyme Mix1 bottle (Component B1) and mix well.

2. Add 100 μL of ddH2O into one Enzyme Mix2 vial (Component B2) and mix well.

3. Transfer 25 μL of 200X Amplite™ Red stock solution, 50 μL of 100X OAC stock solution, and entire vial (100 μL) of Enzyme Mix2 into the Enzyme Mix1 bottle and mix well to make Oxaloacetate working solution. Note: This Oxaloacetate working solution is not stable, use it promptly, and avoid direct exposure to light.

Sample experimental protocol

Table 1. Layout of Oxaloacetate standards and test samples in a solid black 96-well microplate. SD=Oxaloacelate standard (SD1 - SD7, 0.1 to 100 µM), BL=Blank Control, TS=Test Samples. 

BL BL TS TS
SD1 SD1 ... ...
SD2 SD2 ... ...
SD3 SD3    
SD4 SD4    
SD5 SD5    
SD6 SD6    
SD7 SD7    

Table 2. Reagent composition for each well.

Well Volume Reagent
SD1 - SD7 50 µL Serial Dilutions (0.1 to 100 µM)
BL 50 µL Assay Buffer (Component C)
TS 50 µL test sample
  1. Prepare Oxaloacetate standards (SD), 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 Oxaloacetate working solution to each well of Oxaloacetate standard, blank control, and test samples to make the total Oxaloacetate assay volume of 100 µL/well. For a 384-well plate, add 25 µL of Oxaloacetate working solution into each well instead, for a total volume of 50 µL/well. Note: Run the Oxaloacetate assay at pH 6.5 to 7.0.

  3. Incubate the reaction at room temperature for 30 - 60 minutes, protected from light.

  4. Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 540/590 nm (Cutoff = 570 nm).
Example data analysis and figures

The reading (RFU) obtained from the blank standard well is used as a negative control. Subtract this value from the other standards' readings to obtain the base-line corrected values. Then, plot the standards' readings to obtain a standard curve and equation. This equation can be used to calculate Oxaloacetate samples. We recommend using the Online Linear Regression Calculator which can be found at:

https://www.aatbio.com/tools/linear-logarithmic-semi-log-regression-online-calculator

Figure 1. Oxaloacetate dose response was measured with the Amplite™ Fluorimetric Oxaloacetate Assay Kit on a solid black 96-well plate using a Gemini microplate reader (Molecular Devices).

Disclaimer
AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email info@aatbio.com if you have any questions.





References & Citations

Enzymatic assay for D-aspartic acid using D-aspartate oxidase and oxaloacetate decarboxylase
Authors: Kato S, Ikuta T, Hemmi H, Takahashi S, Kera Y, Yoshimura T.
Journal: Biosci Biotechnol Biochem (2012): 2150

Assay of blood and tissue oxaloacetate and alpha-ketoglutarate by isotope dilution gas chromatography-mass spectrometry
Authors: Laplante A, Comte B, Des Rosiers C.
Journal: Anal Biochem (1995): 580

Effect of oxaloacetate and phosphorylation on ATP-citrate lyase activity
Authors: Pentyala SN, Benjamin WB.
Journal: Biochemistry (1995): 10961

Novel oxaloacetate effect on mitochondrial Ca2+ movement
Authors: Leikin YN, Zharova TV, Tjulina OV.
Journal: FEBS Lett (1993): 35

A sensitive multienzymatic assay for the measurement of pyruvate, dihydroxyacetone phosphate, oxaloacetate, and acetoacetate in clear extracts from biological samples
Authors: Arias-Mendoza F, Pina E.
Journal: Prep Biochem (1991): 211

Flow-injection analysis of amino acids and their metabolites by immobilized vitamin B6-dependent enzymes. Sensitive determination of L-aspartate, L-glutamate, 2-oxoglutarate, and oxaloacetate
Authors: Kurkijarvi K, Vierijoki T, Korpela T.
Journal: Ann N Y Acad Sci (1990): 394

Pyruvate dehydrogenase activity in osmotically shocked rat brain mitochondria: stimulation by oxaloacetate
Authors: Haas RH, Thompson G, Morris B, Conright K, Andrews T.
Journal: J Neurochem (1988): 673

Activity of maize leaf phosphoenolpyruvate carboxylase in relation to tautomerization and nonenzymatic decarboxylation of oxaloacetate
Authors: Walker GH, Ku MS, Edwards GE.
Journal: Arch Biochem Biophys (1986): 489

Kinetic mechanism of Escherichia coli isocitrate dehydrogenase and its inhibition by glyoxylate and oxaloacetate
Authors: Nimmo HG.
Journal: Biochem J (1986): 317

The interaction of dithiothreitol and acetyl coenzyme A in a radiochemical assay for rat brain ATP:citrate oxaloacetate lyase
Authors: Simpson J.
Journal: J Neurochem (1981): 100


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Additional Documents

 
Safety Data Sheet (SDS)


Certificate of Analysis