Amplite® Colorimetric Oxaloacetate Assay Kit Red Color

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® Colorimetric 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 an absorbance microplate reader at 575 nm.

Example protocol

AT A GLANCE

Protocol Summary

Prepare test samples and diluted oxaloacetate standards (50 µL)
Add equal volume of working solution (50 µL)
Incubate at RT for 30 minutes to 1 hour
Monitor absorbance intensity at 575 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. Oxaloacetate standard solution (100 mM)

Add 100 µL of ddH₂O into Oxaloacetate Standard vial (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.

PREPARATION OF STANDARD SOLUTION

For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/13840

Oxaloacetate standard

Add 10 µL of 100 mM oxaloacetate into 990 µL of assay buffer (Component C) to get 1 mM oxaloacetate solution. Take 320 µL of 1 mM oxaloacetate standard solution into 680 µL assay buffer to make 320 µM oxaloacetate solution (OOA7). Then perform 1:2 serial dilutions to get remainder of serially diluted oxaloacetate standards (OOA6 - OOA1).

PREPARATION OF WORKING SOLUTION

Add 50 µL of ddH2O into Oxaloacetate Decarboxcylase (Component D) to make 100X Oxaloacetate Decarboxcylase solution.
Add 5 mL of Assay Buffer (Component C) into one Enzyme Mix 1 bottle (Component B1) and mix well.
Add 100 μL of ddH2O into one Enzyme Mix 2 vial (Component B2) and mix well.
Transfer 25 μL of 200X Amplite™ Red stock solution, 50 μL of OAC stock solution (from 1), and the entire vial (100 μL) of Enzyme Mix 2 (from 3) into the Enzyme Mix 1 bottle and mix well.
Note Avoid direct exposure to light and use promptly.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of oxaloacetate standards and test samples in a clear bottom 96-well microplate. OAA= Oxaloacetate Standard (OOA1 - OOA7, 5 to 320 µM), BL=Blank Control (assay buffer), TS=Test Sample.

BL	BL	TS	TS
OOA1	OOA1	...	...
OOA2	OOA2	...	...
OOA3	OOA3
OOA4	OOA4
OOA5	OOA5
OOA6	OOA6
OOA7	OOA7

Table 2. Reagent composition for each well.

Well	Volume	Reagent
OOA1 - OOA7	50 µL	Serial Dilution (5 to 320 µM)
BL	50 µL	Assay Buffer (Component C)
TS	50 µL	Test Sample

Prepare oxaloacetate standards (OOA), blank controls (BL), and test samples (TS) according to the layout provided in Table 1 and Table 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.
Add 50 µL of assay working solution into 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 assay 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.
Incubate the reaction at room temperature for 30 minutes to 1 hour.
Monitor the absorbance increase with an absorbance plate reader at 575 nm.

References

View all 17 references: Citation Explorer

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

Page updated on December 17, 2024

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