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Amplite® Colorimetric Pyruvate Assay Kit
Pyruvate is an important chemical compound in intracellular metabolic pathways. It is derived from metabolism of glucose known as glycolysis. One molecule of glucose breaks down into two molecules of pyruvate, which supplies living cells energy through one of two ways. When oxygen is present (aerobic respiration), pyruvate is converted into acetyl-CoA by pyruvate dehydrogenase which enters citric acid cycles (also known as the Krebs cycle) to generate ATP. When there is insufficient oxygen is available, the pyruvate is broken down anaerobically, creating lactate in animals and ethanol in plants and microorganisms. Abnormal levels of pyruvate, or concentration ratio of lactate-to-pyruvate may be linked to liver disease or metabolic disorders and it is a diagnostic measurement in patient's clinical and other laboratory studies. AAT Bioquest's Amplite® Fluorimetric Pyruvate Assay Kit offers a sensitive fluorescent assay for quantifying pyruvate in biological samples. It utilizes an enzyme coupled reaction that releases hydrogen peroxide, which can be detected by pyruvate sensor in a fluorescence microplate reader.
Example protocol
AT A GLANCE
Protocol Summary
- Prepare test samples along with serially diluted pyruvate standards (50 µL)
- Add equal volume of working solution (50 µL)
- Incubate at room temperature for 30 minutes to 1 hour
- Monitor absorbance intensity at 575 nm
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.
Quest Fluor™ Pyruvate Sensor stock solution (200X)
Add 50 µL of DMSO (Component E) into Quest Fluor™ Pyruvate Sensor (Component A) to make 200X Quest Fluor™ Pyruvate Sensor stock solution.PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/13821
https://www.aatbio.com/tools/serial-dilution/13821
Pyruvate standard
Add 2 μL of 100 mM Pyruvate (Component D) into 998 μL of PBS (pH 7.0) to have 200 µM Pyruvate standard solution (PS7). And then perform 1:2 serial dilutions to get remaining serially diluted pyruvate standards (PS6 - PS1).PREPARATION OF WORKING SOLUTION
Add 5 mL Assay Buffer (Component C) into one Enzyme Mix1 bottle (Component B1) and mix well. Add 100 μL of ddH2O into one Enzyme Mix2 vial (Component B2) and mix well. Transfer entire vial of Enzyme Mix2 (100 μL) and 25 uL of 200X pyruvate sensor stock solution into the Enzyme Mix1 bottle and mix well.
Note The working solution is not stable. Use promptly and avoid direct exposure to light.
Note The working solution is not stable. Use promptly and avoid direct exposure to light.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of pyruvate standards and test samples in a white clear 96-well microplate. PS=Pyruvate Standard (PS1 - PS7, 3.125 to 200 µM), BL=Blank Control (PBS), TS=Test Sample.
Table 2. Reagent composition for each well.
| BL | BL | TS | TS |
| PS1 | PS1 | ... | ... |
| PS2 | PS2 | ... | ... |
| PS3 | PS3 | ||
| PS4 | PS4 | ||
| PS5 | PS5 | ||
| PS6 | PS6 | ||
| PS7 | PS7 |
| Well | Volume | Reagent |
| PS1 - PS7 | 50 µL | Serial Dilutions (3.125 to 200 µM) |
| BL | 50 µL | PBS |
| TS | 50 µL | test sample |
- Prepare pyruvate standards (PS), 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 working solution to each well of pyruvate standard, blank control, and test samples to make the total pyruvate assay volume of 100 µL/well. For a 384-well plate, add 25 µL of working solution into each well instead, for a total volume of 50 µL/well.
Note Run the pyruvate assay at pH 6.5 to 7.0. - Incubate the reaction mixture at room temperature for 30 minutes to 1 hour.
- Monitor the absorbance increase with an absorbance microplate reader at 575 nm.
Citations
View all 1 citations: Citation Explorer
Biochemical and Enzymatic Analyses to Understand the Accumulation of $\gamma$-Aminobutyric Acid in Wheat Grown under Flooding Stress
Authors: Komatsu, Setsuko and Nishiyama, Natsuru and Diniyah, Azzahrah
Journal: Oxygen (2023): 120--132
Authors: Komatsu, Setsuko and Nishiyama, Natsuru and Diniyah, Azzahrah
Journal: Oxygen (2023): 120--132
References
View all 51 references: Citation Explorer
A replaceable dual-enzyme capillary microreactor using magnetic beads and its application for simultaneous detection of acetaldehyde and pyruvate
Authors: Shi J, Zhao W, Chen Y, Guo L, Yang L.
Journal: Electrophoresis (2012): 2145
Authors: Shi J, Zhao W, Chen Y, Guo L, Yang L.
Journal: Electrophoresis (2012): 2145
Detection of inflammatory arthritis by using hyperpolarized 13C-pyruvate with MR imaging and spectroscopy
Authors: MacKenzie JD, Yen YF, Mayer D, Tropp JS, Hurd RE, Spielman DM.
Journal: Radiology (2011): 414
Authors: MacKenzie JD, Yen YF, Mayer D, Tropp JS, Hurd RE, Spielman DM.
Journal: Radiology (2011): 414
Amplex UltraRed enhances the sensitivity of fluorimetric pyruvate detection
Authors: Zhu A, Romero R, Petty HR.
Journal: Anal Biochem (2010): 123
Authors: Zhu A, Romero R, Petty HR.
Journal: Anal Biochem (2010): 123
Comparison of high-performance liquid chromatography with fluorescence detection versus enzymatic assay to measure blood pyruvate in clinical practice
Authors: Wolff F, El Khattabi C, Bourdon F, Willems D.
Journal: Clin Biochem (2009): 1099
Authors: Wolff F, El Khattabi C, Bourdon F, Willems D.
Journal: Clin Biochem (2009): 1099
Faecal tumour pyruvate kinase M2: not a good marker for the detection of colorectal adenomas
Authors: Shastri YM, Stein JM.
Journal: Br J Cancer (2008): 1366; author reply 1367
Authors: Shastri YM, Stein JM.
Journal: Br J Cancer (2008): 1366; author reply 1367
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