Measuring Proteasome Activity in Different Subcellular Compartments of the Rat Brain

Begin with subcellular nuclear and synaptic fractions collected from the lateral amygdala of the brains of both control and fear-conditioned rats.

Each subcellular fraction contains varying concentrations of 20S proteasomes, the catalytic core of the proteasome complex responsible for degrading unwanted cellular proteins.

Add an assay buffer containing ATP and a fluorogenic peptide substrate to the samples and incubate.

In the presence of ATP, the proteasome cleaves the fluorogenic peptide, releasing free fluorogenic molecules.

Using a plate reader, measure the fluorescence intensity at regular intervals.

Quantify the fluorescence by comparing it to known standard concentrations of the fluorogenic molecule.

The fluorescence intensity is proportional to the proteasome activity in the sample.

Increased proteasome activity in the nuclear fraction of fear-conditioned rats suggests changes in memory-related gene expression, while decreased activity in the synaptic fraction indicates changes in synaptic protein expression necessary for long-term memory maintenance.

To set up the assay, pre-warm the plate reader to 37 degrees Celcius and hold through the run. Set the Excitation to 360 nanometers and Emission to 460 nanometers. If the 96 well plate used is clear, set the Optics Position to Bottom. If a dark 96-well plate is used, Set Optics position to Top. Program a kinetic run with the time of 2 hours, scanning every 30 minutes.

Reconstitute the 20s 10x assay buffer provided in the kit with 13.5 milliliters of ultrapure water. Add 14 microliters of 100 millimolar ATP to the now 1x buffer. This significantly enhances proteasome activity in the samples and improves assay reliability. Reconstitute the AMC standard provided in the kit with 100 microliters of DMSO. Perform steps using the AMC standard in the dark or under low light conditions, as the standard is light-sensitive.

Create a standard curve of AMC from a series of high-to-low AMC concentrations. This curve will be used for plate-reader calibration and analysis of proteasome activity in the homogenized samples. Reconstitute the proteasome substrate provided in the kit with 65 microliters of DMSO. Also perform this step in the dark or under low light conditions, as the substrate is light sensitive.

Then create a 1-to-20 dilution of the proteasome substrate in a new 1.5 milliliters microcentrifuge tube, using 20 S assay buffer. Add a normalized amount of the desired samples to a 96-well plate. Run each sample in duplicate. The amount of sample needed varies based on tissue preparation. Generally, 10 to 20 micrograms is sufficient for any subcellular fraction.

Bring the sample well volume to 80 microliters with ultrapure water. The amount added depends on the volume of sample added. In two separate wells, add 80 microliters of water alone. These will be the assay blanks. Add 10 microliters of 20 S assay buffer to each well, including assay blanks. Use a repeater or automated pipette to ensure consistent assay volume across the wells.

Turn off the lights or enter a dark room. Add all 100 microliters of diluted AMC standards to a new well, using a single well for each standard. In the dark, use a repeater or automated pipette to add 10 microliters of diluted proteasome substrate to wells containing sample and assay blanks, but not the AMC standard. Place the plate into the plate reader and start the kinetic run.