Amplite® Fluorimetric Proteasome 20S Activity Assay Kit *Green Fluorescence*
Example protocol
AT A GLANCE
- Prepare cells with test compounds (100 µL/well/96-well plate or 25 µL/well/384-well plate)
- Add equal volume of Proteasome working solution (100 µL/well/96-well plate or 25 µL/well/384-well plate)
Incubate at 37°C for at least 1 hour
- Monitor fluorescence intensity at Ex/Em = 490/525 nm (Cutoff = 515 nm)
Thaw all the kit components at room temperature before use.
CELL PREPARATION
For guidelines on cell sample preparation, please visit https://www.aatbio.com/resources/guides/cell-sample-preparation.html
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 25 µL of DMSO (Component C) to the vial of Proteasome LLVY-R110 Substrate (Component A), and mix well to make 400X Proteasome LLVY-R110 Substrate stock solution.
PREPARATION OF WORKING SOLUTION
Add 25 μL of 400X Proteasome LLVY-R110 Substrate stock solution into 10 mL of Assay Buffer (Component B) and mix well to make Proteasome working solution. Note: This Proteasome working solution is enough for 1 plate. Protect from light.
SAMPLE EXPERIMENTAL PROTOCOL
- Treat cells with 10 µL of 10X test compound (for a 96-well plate) or 5 µL of 5X test compound (for a 384-well plate) in PBS or desired buffer. For blank wells (medium without the cells), add the corresponding amount of compound buffer.
Incubate the cell plates in a 5% CO2, 37°C incubator for a desired period of time. Note: Pure proteasome or cell lysates can be used directly for screening the proteasome inhibitors.
- Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Proteasome working solution.
Incubate the plate at 37°C for at least 1 hour (2 hours to overnight), protected from light. Note: Each cell line should be evaluated on an individual basis to determine the optimal incubation time.
- Monitor the fluorescence intensity (top read) at Ex/Em = 490/525 nm (Cutoff = 515 nm).
Spectrum
Citations
Authors: Shao, Xinyang and Tian, Meng and Yin, Junlong and Duan, Haifeng and Tian, Ye and Wang, Hui and Xia, Changsheng and Wang, Ziwei and Zhu, Yanxi and Wang, Yifan and others,
Journal: Nature Communications (2024): 8633
Authors: Wang, Yankun and Wang, Chu
Journal: Cell Death \& Differentiation (2022): 1--12
Authors: Guo, Susu and Chen, Yuxin and Xue, Xiangfei and Yang, Yueyue and Wang, Yikun and Qiu, Shiyu and Cui, Jiangtao and Zhang, Xiao and Ma, Lifang and Qiao, Yongxia and others,
Journal: Cell death discovery (2021): 1--13
Authors: Guo, Susu and Chen, Yuxin and Yang, Yueyue and Zhang, Xiao and Ma, Lifang and Xue, Xiangfei and Qiao, Yongxia and Wang, Jiayi
Journal: Cell death \& disease (2021): 1--18
Authors: Zhang, Li and Wei, Peng-Fei and Song, Yong-Hong and Dong, Liang and Wu, Ya-Dong and Hao, Zong-Yao and Fan, Song and Tai, Sheng and Meng, Jia-Lin and Lu, Yang and others, undefined
Journal: Biomaterials (2019): 119248