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PhosphoWorks™ Colorimetric ATP Assay Kit
Example protocol
AT A GLANCE
Protocol summary
- Prepare ATP working solution (50 µL)
- Add ATP standards or test samples (50 µL)
- Incubate at room temperature for 10 - 30 minutes
- Monitor absorbance at 570 nm
Important notes
Thaw the kit components at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
1. AmpliteTM Red Substrate stock solution (200X):
Add 30 µL of DMSO (Component E) into the vial of AmpliteTM Red Substrate (Component A) to make 200X AmpliteTM Red Substrate stock solution.
2. ATP standard solution (10 mM):
Add 0.5 mL of ddH2O into the vial of ATP Standard (Component D) to make 10 mM ATP standard solution.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/21617
Add 10 µL of 10 mM ATP standard solution into 990 µL 1X PBS buffer to generate 100 µM ATP standard solution (AS7). Take 100 µM ATP standard solution (AS7) and perform 1:2 serial dilutions to get serially diluted ATP standards (AS6-AS1) with 1X PBS buffer.
PREPARATION OF WORKING SOLUTION
1. Add 5 mL of Assay Buffer (Component C) into Enzyme Mix bottle (Component B), and mix well.
2. Add 25 µL of 200X AmpliteTM Red Substrate stock solution to the Enzyme Mix bottle, and mix well to make ATP working solution.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of ATP standards and test samples in a clear bottom 96-well microplate. AS= ATP Standards (AS1 - AS7, 1.56 to 100 µM), BL=Blank Control, TS=Test Samples.
| BL | BL | TS | TS |
| AS1 | AS1 | ... | ... |
| AS2 | AS2 | ... | ... |
| AS3 | AS3 | ||
| AS4 | AS4 | ||
| AS5 | AS5 | ||
| AS6 | AS6 | ||
| AS7 | AS7 |
Table 2. Reagent composition for each well.
| Well | Volume | Reagent |
| AS1 - AS7 | 50 µL | Serial Dilutions (1.56 to 100 µM) |
| BL | 50 µL | 1 X PBS Buffer |
| TS | 50 µL | Test Sample |
- Prepare ATP standards (AS), 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 ATP working solution to each well of ATP standard, blank control, and test samples to make the total ATP assay volume of 100 µL/well. For a 384-well plate, add 25 µL of ATP working solution into each well instead, for a total volume of 50 µL/well.
- Incubate the reaction at room temperature for 10 - 30 minutes, protected from light.
- Monitor the absorbance increase with an absorbance plate reader at OD of 570 nm.
Citations
Authors: Zhang, Miaomiao and Gao, Chenying and Wang, Pengcheng and Li, Rui and Huang, Yiming and Lv, Fengting and Li, Cheng and Bai, Haotian and Zhang, Deqing and Wang, Shu
Journal: ACS nano (2025)
Authors: Alexandrou, Aris T and Duan, Yixin and Xu, Shanxiu and Tepper, Clifford and Fan, Ming and Tang, Jason and Berg, Jonathan and Basheer, Wassim and Valicenti, Tyler and Wilson, Paul F and others,
Journal: Iscience (2022): 105546
Authors: Nowak, E., Kammerer, S., Kupper, J. H.
Journal: Clin Hemorheol Microcirc (2018): 327-336
Authors: Tahara, H., Matsuda, S., Yamamoto, Y., Yoshizawa, H., Fujita, M., Katsuoka, Y., Kasahara, T.
Journal: J Pharmacol Toxicol Methods (2017): 92-99
Authors: Wang, Mengqiao and Xu, Miao and Long, Yan and Fargue, Sonia and Southall, Noel and Hu, Xin and McKew, John C and Danpure, Christopher J and Zheng, Wei
Journal: Scientific Reports (2016)
Safety data sheet