Amplite® Colorimetric Pyruvate Assay Kit

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Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
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H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
UNSPSC	12352200

Overview SDS Protocol

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.

Platform

Absorbance microplate reader

Absorbance	575 nm
Recommended plate	Clear bottom

Components

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

Important Thaw kit components at room temperature before use.

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

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.

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.

BL	BL	TS	TS
PS1	PS1	...	...
PS2	PS2	...	...
PS3	PS3
PS4	PS4
PS5	PS5
PS6	PS6
PS7	PS7

Table 2. Reagent composition for each well.

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.

Images

Figure 1. Pyruvate dose response was measured with the Amplite® Colorimetric Pyruvate Assay Kit on a white clear 96-well plate using a SpectraMax microplate reader (Molecular Devices).

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

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

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