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Amplite® Fluorimetric Proteasome 20S Activity Assay Kit *Green Fluorescence*

Our Amplite® Fluorometric 20S Proteasome Assay Kit is a homogeneous fluorescent assay that measures the chymotrypsin-like protease activity associated with the proteasome complex either in cultured cells or cell lysates. This kit uses LLVY-R110 as a fluorogenic indicator for proteasome activity. Cleavage of LLVY-R110 by proteasome generates the strongly green fluorescent R110 that is monitored fluorimetrically at 520-530 nm with excitation of 480-500 nm. The kit provides all the essential components with an optimized assay protocol. The assay is robust, and can be readily adapted for high-throughput assays for evaluation of proteasome activity or inhibitor screening in cultured cells or in solution. The assay can be performed in a convenient 96-well and 384-well fluorescence microtiter-plate format. The main function of the proteasome is to degrade unneeded or damaged proteins by proteolysis, a chemical reaction that breaks peptide bonds. The proteasomal degradation pathway is essential for many cellular processes, including the cell cycle, the regulation of gene expression, and responses to oxidative stress. The most common form of the proteasome in this pathway is the 26S proteasome, an ATP-dependent proteolytic complex, which contains one 20S (700-kDa) core particle structure and two 19S (700-kDa) regulatory caps. The 20S core contains three major proteolytic activities including chymotrypsin-like, trypsin-like and caspase-like. It is responsible for the breakdown of key proteins involved with apoptosis, DNA repair, endocytosis, and cell cycle control.

Example protocol

AT A GLANCE

Protocol Summary
  1. Prepare cells with test compounds (100 µL/well/96-well plate or 25 µL/well/384-well plate)
  2. Add equal volume of Proteasome working solution (100 µL/well/96-well plate or 25 µL/well/384-well plate)
  3. Incubate at 37°C for at least 1 hour

  4. Monitor fluorescence intensity at Ex/Em = 490/525 nm (Cutoff = 515 nm)
Important Note

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

Proteasome LLVY-R110 Substrate stock solution (400X)

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

  1. 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.
  2. 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.

  3. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Proteasome working solution.
  4. 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.

  5. Monitor the fluorescence intensity (top read) at Ex/Em = 490/525 nm (Cutoff = 515 nm).

Spectrum

Citations

View all 14 citations: Citation Explorer
Biofunctionalized dissolvable hydrogel microbeads enable efficient characterization of native protein complexes
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
Quantitative reactive cysteinome profiling reveals a functional link between ferroptosis and proteasome-mediated degradation
Authors: Wang, Yankun and Wang, Chu
Journal: Cell Death \& Differentiation (2022): 1--12
TRIB2 desensitizes ferroptosis via $\beta$TrCP-mediated TFRC ubiquitiantion in liver cancer cells
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
TRIB2 modulates proteasome function to reduce ubiquitin stability and protect liver cancer cells against oxidative stress
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
MnFe2O4 nanoparticles accelerate the clearance of mutant huntingtin selectively through ubiquitin-proteasome system
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
Page updated on December 6, 2024

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Catalog Number13456
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Spectral properties

Extinction coefficient (cm -1 M -1)

80000

Excitation (nm)

500

Emission (nm)

522

Storage, safety and handling

Certificate of OriginDownload PDF
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

Platform

Fluorescence microplate reader

Excitation490 nm
Emission525 nm
Cutoff515 nm
Recommended plateSolid black
Instrument specification(s)Top read mode

Components

Detection of proteasome activity in Jurkat cells with Amplite® Fluorimetric Proteasome 20S Activity Assay Kit. Jurkat cells were seeded on the same day at 500,000 cells/90 &micro;L/well in a 96-well black wall/clear bottom Costar plate. The cells were treated with or without 50 mM H<sub>2</sub>O<sub>2</sub> for 30 minutes. The proteasome assay loading solution (100 &micro;L/well) was added and incubated in a 5% CO2, 37 &deg;C incubator for 3 hours. The fluorescence intensity was measured at Ex/Em = 490/525 by using a Gemini fluorescent microplate reader (Molecular Devices).
Detection of proteasome activity in Jurkat cells with Amplite® Fluorimetric Proteasome 20S Activity Assay Kit. Jurkat cells were seeded on the same day at 500,000 cells/90 &micro;L/well in a 96-well black wall/clear bottom Costar plate. The cells were treated with or without 50 mM H<sub>2</sub>O<sub>2</sub> for 30 minutes. The proteasome assay loading solution (100 &micro;L/well) was added and incubated in a 5% CO2, 37 &deg;C incubator for 3 hours. The fluorescence intensity was measured at Ex/Em = 490/525 by using a Gemini fluorescent microplate reader (Molecular Devices).
Detection of proteasome activity in Jurkat cells with Amplite® Fluorimetric Proteasome 20S Activity Assay Kit. Jurkat cells were seeded on the same day at 500,000 cells/90 &micro;L/well in a 96-well black wall/clear bottom Costar plate. The cells were treated with or without 50 mM H<sub>2</sub>O<sub>2</sub> for 30 minutes. The proteasome assay loading solution (100 &micro;L/well) was added and incubated in a 5% CO2, 37 &deg;C incubator for 3 hours. The fluorescence intensity was measured at Ex/Em = 490/525 by using a Gemini fluorescent microplate reader (Molecular Devices).