AAT Bioquest

Amplite® Fluorimetric Pyruvate Assay Kit

Pyruvate dose response was measured with the Amplite® Fluorimetric Pyruvate Assay Kit on a solid black 96-well plate using a Gemini microplate reader (Molecular Devices).
Pyruvate dose response was measured with the Amplite® Fluorimetric Pyruvate Assay Kit on a solid black 96-well plate using a Gemini microplate reader (Molecular Devices).
Pyruvate dose response was measured with the Amplite® Fluorimetric Pyruvate Assay Kit on a solid black 96-well plate using a Gemini microplate reader (Molecular Devices).
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Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22


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® Colorimetric Pyruvate Assay Kit offers a sensitive colorimetric assay for quantifying pyruvate in the samples. It utilizes an enzyme coupled reaction that releases hydrogen peroxide, which can be detected by pyruvate sensor by an absorbance microplate reader at 575 nm.


Fluorescence microplate reader

Excitation540 nm
Emission590 nm
Cutoff570 nm
Recommended plateSolid black


Example protocol


Protocol summary

  1. Prepare test samples (50 µL) along with serially diluted pyruvate standards (50 µL)
  2. Add equal volume of pyruvate working solution (50 µL)
  3. Incubate at room temperature for 30 minutes to 1 hour
  4. Monitor fluorescence intensity at Ex/Em = 540/590 nm

Important notes
To achieve the best results, it’s strongly recommended to use the black plates. Thaw kit components at room temperature before use.


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. Quest Fluor™ Pyruvate sensor stock solution (200X):
Add 55 µL of DMSO (Component E) into Quest Fluor™ Pyruvate Sensor (Component A) to make 200X Quest Fluor™ Pyruvate sensor stock solution.

2. Pyruvate standard solution (1 mM):
Add 10 µL of 100 mM Pyruvate (Component D) into 990 µL of PBS (pH 7) to get 1 mM pyruvate solution.


Pyruvate standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/13820

Take 100 µL of 1 mM pyruvate standard solution into 900 µL PBS to make 100 µM pyruvate solution (PS7). And then perform 1:3 serial dilutions to get serially diluted pyruvate standards (PS6 - PS1).


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

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

3. Transfer entire vial (100 μL) of Enzyme Mix 2 and 25 uL of 200X Quest Fluor™ Pyruvate Sensor stock solution into the Enzyme Mix 1 bottle. Mix well. Note: The pyruvate working solution is not stable and should be used promptly. Avoid light.


Table 1. Layout of pyruvate standards and test samples in a solid black 96-well microplate. PS = pyruvate standard (PS1 - PS7, 0.1 to 100 µM); BL = blank control; TS = test sample.


Table 2. Reagent composition for each well.

PS1 - PS750 µLserial dilution (0.1 to 100 µM)
TS50 µLsample
  1. Prepare pyruate standards (PS), blank controls (BL), and test samples (TS) into a solid black 96-well microplate 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.

  2. Add 50 µL of pyruvate working solution into 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. Run the pyruate assay at a pH of 6.5 to 7.0.

  3. Incubate the reaction mixture at room temperature for 30 minutes to 1 hour.

  4. Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 540/590 nm (cut off: 570 nm).



View all 2 citations: Citation Explorer
Interference of reversible redox compounds in enzyme catalysed assays--Electrochemical limitations
Authors: McBeth, Craig and Stott-Marshall, Robert J
Journal: Analytical Biochemistry (2022): 114972
Inhibition of the Monocarboxylate Transporter 1 (MCT1) Promotes 3T3-L1 Adipocyte Proliferation and Enhances Insulin Sensitivity
Authors: Bailey, Tracey and Nieto, Ainhoa and McDonald, Patricia
Journal: International Journal of Molecular Sciences (2022): 1901


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
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
Amplex UltraRed enhances the sensitivity of fluorimetric pyruvate detection
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
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
Sensitivity and specificity of faecal tumour M2 pyruvate kinase for detection of colorectal adenomas in a large screening study
Authors: Haug U, Hundt S, Brenner H.
Journal: Br J Cancer (2008): 133
Hyperpolarized 13C lactate, pyruvate, and alanine: noninvasive biomarkers for prostate cancer detection and grading
Authors: Albers MJ, Bok R, Chen AP, Cunningham CH, Zierhut ML, Zhang VY, Kohler SJ, Tropp J, Hurd RE, Yen YF, Nelson SJ, Vigneron DB, Kurhanewicz J.
Journal: Cancer Res (2008): 8607
Tumor M2-pyruvate kinase in lung cancer patients: immunohistochemical detection and disease monitoring
Authors: Schneider J, Neu K, Grimm H, Velcovsky HG, Weisse G, Eigenbrodt E.
Journal: Anticancer Res (2002): 311
Detection of pyruvate dehydrogenase E1 alpha-subunit deficiencies in females by immunohistochemical demonstration of mosaicism in cultured fibroblasts
Authors: Lib MY, Brown RM, Brown GK, Marusich MF, Capaldi RA.
Journal: J Histochem Cytochem (2002): 877
Measurement of pyruvate in blood by high-performance liquid chromatography with fluorescence detection
Authors: Wulkan RW, Verwers R, Neele M, Mantel MJ.
Journal: Ann Clin Biochem (2001): 554