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

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)

Thaw all the kit components at room temperature before use.

For guidelines on cell sample preparation, please visit https://www.aatbio.com/resources/guides/cell-sample-preparation.html

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.

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.

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).