Amplite® Fluorimetric Proteasome 20S Activity Assay Kit *Green Fluorescence*
![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 µL/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 µL/well) was added and incubated in a 5% CO2, 37 °C incubator for 3 hours. The fluorescence intensity was measured at Ex/Em = 490/525 by using a Gemini fluorescent microplate reader (Molecular Devices).](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Famplite-fluorimetric-proteasome-20s-activity-assay-kit-green-fluorescence%2Ffigure-for-amplite-fluorimetric-proteasome-20s-activity-assay-kit-green-fluorescence_ryTKD.jpg&w=640&q=75)
![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 µL/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 µL/well) was added and incubated in a 5% CO2, 37 °C incubator for 3 hours. The fluorescence intensity was measured at Ex/Em = 490/525 by using a Gemini fluorescent microplate reader (Molecular Devices).](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Famplite-fluorimetric-proteasome-20s-activity-assay-kit-green-fluorescence%2Ffigure-for-amplite-fluorimetric-proteasome-20s-activity-assay-kit-green-fluorescence_ryTKD.jpg&w=640&q=75)
![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 µL/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 µL/well) was added and incubated in a 5% CO2, 37 °C incubator for 3 hours. The fluorescence intensity was measured at Ex/Em = 490/525 by using a Gemini fluorescent microplate reader (Molecular Devices).](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Famplite-fluorimetric-proteasome-20s-activity-assay-kit-green-fluorescence%2Ffigure-for-amplite-fluorimetric-proteasome-20s-activity-assay-kit-green-fluorescence_ryTKD.jpg&w=128&q=25)
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).
Authors: Wang, Yankun and Wang, Chu
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