JoVE Encyclopedia of Experiments
Neuroscience
0 views • 4:10 min • July 8th, 2025
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 nan
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