Calculator
Amplite® Fluorimetric Pyruvate Assay Kit
Example protocol
AT A GLANCE
Protocol summary
- Prepare test samples (50 µL) along with serially diluted pyruvate standards (50 µL)
- Add equal volume of pyruvate working solution (50 µL)
- Incubate at room temperature for 30 minutes to 1 hour
- 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.
PREPARATION OF STOCK SOLUTION
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.
PREPARATION OF STANDARD SOLUTION
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).
PREPARATION OF WORKING SOLUTION
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.
SAMPLE EXPERIMENTAL PROTOCOL
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.
| 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 dilution (0.1 to 100 µM) |
| BL | 50 µL | PBS |
| TS | 50 µL | sample |
- 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.
- 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.
- Incubate the reaction mixture at room temperature for 30 minutes to 1 hour.
- Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 540/590 nm (cut off: 570 nm).
Citations
Authors: Bailey, Tracey and Nieto, Ainhoa and McDonald, Patricia
Journal: International Journal of Molecular Sciences (2022): 1901
Authors: McBeth, Craig and Stott-Marshall, Robert J
Journal: Analytical Biochemistry (2022): 114972
References
Authors: Shi J, Zhao W, Chen Y, Guo L, Yang L.
Journal: Electrophoresis (2012): 2145
Authors: MacKenzie JD, Yen YF, Mayer D, Tropp JS, Hurd RE, Spielman DM.
Journal: Radiology (2011): 414
Authors: Zhu A, Romero R, Petty HR.
Journal: Anal Biochem (2010): 123
Authors: Wolff F, El Khattabi C, Bourdon F, Willems D.
Journal: Clin Biochem (2009): 1099
Authors: Shastri YM, Stein JM.
Journal: Br J Cancer (2008): 1366; author reply 1367
Safety data sheet