Measuring Proteasome Activity in Different Subcellular Compartments of the Rat Brain

1. Proteasome Activity Assay

NOTE: Proteasome activity can be measured in homogenized brain tissue using a slightly modified version of the 20S Proteasome Activity Kit. This assay does not directly measure the activity of complete 26S proteasome complexes. Rather, it measures the activity of the 20S core, meaning it can only serve as a proxy to understand the activity of the core itself as opposed to the entire 26S proteasome complex. The success of this assay declines with repeated freeze-thaw cycles and/or increasing levels of detergents, particularly ionic, and requires the use of a plate reader with a 360/460 (excitation/emission) filter set and heating capabilities up to 37 °C.

1. Plate reader settings: Pre-warm to 37 °C and hold through the run.​
   1. Set excitation to 360 and emission to 460. If the 96 well plate used is clear, set the optics position to Bottom. If a dark/black 96 well plate is used, set optics position to Top.
   2. Set older plate reader models to Auto-Gain under the fluorescent read options; newer models are preset for this. Program a kinetic run with a time of 2 h, scanning (reading) every 30 min.
2. Reconstitute the 10x assay buffer provided in the kit with 13.5 mL of ultrapure water. Add 14 µL of 100 mM ATP to the now 1x buffer; this significantly enhances proteasome activity in the samples and improves assay reliability. The final 20S Assay buffer can be stored on ice or at 4 °C until needed and is stable for several months.
3. Reconstitute the AMC standard provided in kit with 100 µL of DMSO. Perform this step in the dark or under low light conditions, as the standard is light sensitive.
4. Create a standard curve of AMC using the reconstituted standard, in the dark or under low light conditions.
   1. In separate 0.5 mL microcentrifuge tubes, add 16, 8, 6.4, 3.2, 1.6, 0.8, 0.4 and 0 µL of the AMC standard, which corresponds to 20, 10, 8, 4, 2, 1, 0.5 and 0 µM AMC concentration.
   2. To these tubes, in the same order, add 84, 92, 93.6, 96.8, 98.4, 99.2, 99.6 and 100 µL of 20S Assay buffer. This creates a series of high to low AMC concentrations, which will be used for plate reader calibration and analysis of proteasome activity in the homogenized samples.
   3. Store all diluted standards on ice in the dark until needed.
5. Reconstitute the proteasome substrate (Suc-LLVY-AMC) provided in kit with 65 µL of DMSO. Perform this step in the dark or under low light conditions, as the substrate is light sensitive. Create a 1:20 dilution of the proteasome substrate in a new 1.5 mL microcentrifuge tube using 20S Assay buffer. Store the diluted substrate on ice in the dark until needed.
   NOTE: For example, if the plate will have 10 samples and 1 blank, you will need enough diluted substrate for 22 wells (with duplicates) at 10 µL per well. This equates to 220 µL need + 30 µL for pipetting errors, requiring 12.5 µL of substrate and 237.5 µL of 20S Assay buffer.
6. Thaw desired samples (if frozen) and add a normalized amount to a 96 well plate. Run each sample in duplicates. The amount of sample needed varies based on tissue preparation. Generally, 10-20 µg is sufficient for any subcellular fraction.
7. Bring the sample well volume to 80 µL with ultrapure water. The amount added depends on the volume of sample added. For example, if sample 1 was 4.5 µL of protein and sample 2 was 8.7 µL, then the amount of water needed will be 75.5 µL and 71.3 µL, respectively. In two separate wells, add 80 µL of water alone; these will be the Assay Blanks.
   NOTE: In order to limit changes in protein volume due to pipetting errors, protein concentrations can be normalized to the least concentrated sample. This will allow use of the same sample volume in all conditions.
8. Add 10 µL of 20S Assay buffer to each well, including Assay Blanks. A repeater/automated pipette is recommended here to ensure consistent assay volume across wells.
9. Optional: At this stage, introduce in vitro manipulations if desired; this will require that each sample has an additional 2 wells per treatment, including the vehicle. If so, add 5-10 µL of drug/compound of interest to 2 of the sample wells and an equivalent volume of control/vehicle to another 2 wells. Place the plate onto the pre-warmed plate reader or into a 37 °C incubator for 30 min.
10. Turn off the lights or enter a dark room. Add all 100 µL of diluted AMC standards to a new well; each standard will have a single well.
11. In the dark, add 10 µL of diluted proteasome substrate to wells containing sample and Assay Blanks, but not to the AMC standard. A repeater/automated pipette is recommended here to ensure consistent assay volume across wells.
12. Place the plate into the plate reader and start the kinetic run.
    NOTE: The plate does not need to be under constant agitation during the kinetic run, however, the user can choose to if desired
13. At end of the kinetic run, export raw 360/460 fluorescent values to Microsoft Excel.
    1. Average together duplicate wells for each standard, each sample and the Assay Blank for all 5 scans. Raw fluorescent values should increase across scans for the samples but remain stable (or decrease slightly) for standards and Assay Blanks.
    2. Take the highest AMC standard well average and divide by the known concentration (20 µM). Divide this value by the sample concentration used in the assay to get standardized AMC value. For each sample and Assay Blank average, divide by the standardized AMC value.
    3. Take this final value and divide it by the sample concentration used in the assay to get the normalized value for each sample and the Assay Blank. Do this for all 5 scans.
    4. Subtract the normalized Assay Blank value from each normalized sample value for all 5 scans.