Materials and Reagents

Cells
HEPES (Sigma-Aldrich, catalog number: H3375 )
MgCl₂ (Sigma-Aldrich, catalog number: M2670 )
EDTA (Sigma-Aldrich, catalog number: E5134 )
EGTA (Sigma-Aldrich, catalog number: E4378 )
Sucrose (MP Biomedicals, catalog number: 821713 )
DTT (Life Technologies, catalog number: 15508013 )
Proteasome substrates [dissolved in DMSO (Sigma-Aldrich, catalog number: D8418 ) to a final concentration of 10 mM and stored frozen at -20 °C]
1. Suc-LLVY-AMC (chymotrypsin-like activity substrate) (Enzo Life Sciences, catalog number: P802 )
2. Z-ARR-AMC (trypsin-like activity substrate) (EMD Millipore, catalog number: 539149 )
3. Z-LLE-AMC (caspase-like activity substrate) (EMD Millipore, catalog number: 539141 )
PBS without Ca²⁺ and Mg²⁺ (Sigma-Aldrich, catalog number: P802)
Coomassie Protein Assay Reagent (Thermo Fisher Scientific, catalog number: 1856209 )
BSA protein standard (Thermo Scientific, catalog number: 23209 )
ATP (Sigma-Aldrich, catalog number: A3377 )
Proteasome Lysis/Assay Buffer (see Recipes)

Equipment

60 mm tissue culture dishes
Rubber policeman or other type of cell scraper
1.7 ml microcentrifuge tube
Black walled 96 well plates (Corning, Costar^®, catalog number: 3603 )
Clear 96 well plates (Greiner Bio-One GmbH, catalog number: 655101 )
Sonicator (GlobalSpec, model: W-380 )
Microcentrifuge
Fluorescent plate reader
Visible plate reader

Procedure

Sample Preparation
1. Rinse cells in 60 mm dishes 2 times with ice cold PBS.
2. Scrape cells using a rubber policeman or other type of cell scraper into 400 µl lysis buffer, transfer to a 1.7 ml microcentrifuge tube and place on ice.
3. Sonicate for 10 sec using microtip set on ~2.
4. Centrifuge at 16,000 x g for 10 min at 4 °C.
5. Transfer supernatant to a fresh 1.7 ml microcentrifuge tube (can store at -70 °C until use in assay).
Assay
1. Prepare assay buffer (make up more than you need; e.g. if you need 3 ml, make 3.5 ml).
2. Add proteasome substrates to assay buffer (2.5 µl/sample; 100 µM final concentration) (make up slightly more than you need; e.g. if you have 12 samples, make up enough for 13).
3. Put 2 x 200 µl aliquots of assay buffer with substrate into 2 wells of a black walled 96 well plate for each sample.
4. Add 50 µl cell lysate to each well (include 2 wells with lysis buffer with no cells as assay blanks).
5. Incubate 60 min at 37 °C in order to allow the cleavage of the fluorophore from the proteasome substrate.
6. Read A₃₆₀ex/A₄₆₀em on fluorescent plate reader.
7. Normalize to protein determined with the Coomassie Protein Assay following the manufacturer’s instructions for a microplate reader and using 5 µl of lysate.

Recipes

Proteasome Lysis/Assay Buffer
50 mM HEPES (pH 7.8)
10 mM NaCl
1.5 mM MgCl₂
1 mM EDTA
1 mM EGTA
250 mM sucrose
Sterile filter and stored at 4 °C
For lysis buffer: add DTT to 5 mM final concentration (use 1 M stock)
For assay buffer: add DTT to 5 mM final concentration and ATP to 2 mM final concentration

Acknowledgments

This protocol was adapted from Valera et al. (2013). The research was supported by grants from NIH (RO1AG035055), the Fritz B. Burns Foundation and the Alzheimer’s Association.

References

Betarbet, R., Sherer, T. B. and Greenamyre, J. T. (2005). Ubiquitin-proteasome system and Parkinson's diseases. Exp Neurol 191 Suppl 1: S17-27.
Chung, K. K., Dawson, V. L. and Dawson, T. M. (2001). The role of the ubiquitin-proteasomal pathway in Parkinson's disease and other neurodegenerative disorders. Trends Neurosci 24(11 Suppl): S7-14.
Ciechanover, A. (2005). Proteolysis: from the lysosome to ubiquitin and the proteasome. Nat Rev Mol Cell Biol 6(1): 79-87.
Ciechanover, A. and Brundin, P. (2003). The ubiquitin proteasome system in neurodegenerative diseases: sometimes the chicken, sometimes the egg. Neuron 40(2): 427-446.
Kisselev, A. F. and Goldberg, A. L. (2001). Proteasome inhibitors: from research tools to drug candidates. Chem Biol 8(8): 739-758.
Valera, E., Dargusch, R., Maher, P. A. and Schubert, D. (2013). Modulation of 5-lipoxygenase in proteotoxicity and Alzheimer's disease. J Neurosci 33(25): 10512-10525.