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Amplite® Fluorimetric Aldehyde Quantitation Kit
Example protocol
AT A GLANCE
- Prepare Aldehyde Standards and/or test samples (50 µL)
- Add AldeLight™ Blue working solution (50 µL)
- Incubate at RT for at least 30 minutes
- Add 25 µL of Reaction Buffer
- Monitor fluorescence increase at Ex/Em = 365/435 nm
Thaw all the kit components to room temperature before starting the experiment.
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
Add 40 µL of DMSO (Component E) into the vial of AldeLight™ Blue (Component A) to make 250X AldeLight™ Blue stock solution.
Add 1 mL of ddH2O into the vial of Aldehyde Standard (Component D) to make a 10 mM aldehyde standard solution.
PREPARATION OF STANDARD SOLUTIONS
https://www.aatbio.com/tools/serial-dilution/10052
PREPARATION OF WORKING SOLUTION
Add 20 μL of 250X AldeLight™ Blue stock solution into 5 mL of Assay Buffer (Component B), and mix well to make AldeLight™ Blue working solution.
Note: 5 mL of AldeLight™ Blue working solution is enough for one plate. The reaction mixture is not stable, and best used within 2 hours.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of Aldehyde Standards and test samples in a solid black 96-well microplate. AS= Aldehyde Standards (AS1 - AS7, 1 to 1000 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 Dilution (1 to 1000 µM) |
| BL | 50 µL | ddH2O |
| TS | 50 µL | test sample |
Prepare Aldehyde 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.
- Optional:The sensor will react with the thiol, so treat samples (including the control) with 2-5 mM H2O2 for 30 min if the sample contains thiol prior to addition of AldeLight™ Blue.
Add 50 µL of AldeLight™ Blue working solution to each well of aldehyde standard, blank control, and test samples to make the total aldehyde assay volume of 100 µL/well. For a 384-well plate, add 25 µL of AldeLight™ Blue working solution into each well instead, for a total volume of 50 µL/well.
Incubate the reaction mixture at room temperature for 30 minutes or more, protected from light.
Add 25 µL of Reaction Buffer (Component C) into each well.
Monitor the fluorescence increase at Ex/Em = 365/435 nm using a fluorescence plate reader.
Citations
Authors: Cao, Xiufei and Guo, Huixing and Dai, Yongjun and Jiang, Guangzhen and Liu, Wenbin and Li, Xiangfei and Zhang, Dingdong and Huang, Yangyang and Wang, Xi and Hua, Haokun and others,
Journal: Redox Biology (2024): 103096
Authors: Zhou, Junmei and Sun, Chunbao and Yang, Lu and Wang, Jinhui and Jn-Simon, Natacha and Zhou, Chen and Bryant, Andrew and Cao, Qi and Li, Chenglong and Petersen, Bryon and others,
Journal: The FASEB Journal (2022): e22224
Authors: Liu, Baoshan and Wang, Jiali and Li, Minghua and Yuan, Qiuhuan and Xue, Mengyang and Xu, Feng and Chen, Yuguo
Journal: Oncotarget (2017): 19413
Authors: Kumar, Sudhir and Wang, Jiang and Rani, Richa and G, undefined and hi, Ch and rashekhar R, undefined
Journal: PloS one (2016): e0147864
Authors: Lin, Chun-Jui and Kuan, Chen-Hsiang and Wang, Li-Wen and Wu, Hsi-Chin and Chen, Yunching and Chang, Chien-Wen and Huang, Rih-Yang and Wang, Tzu-Wei
Journal: Biomaterials (2016): 12--26
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