Determination of Proteasomal Activities - Protein Misfolding and Cellular Stress in Disease and Aging - page 179

2. Materials

2.1. Buffers and Stock
Solutions

1. Proteasome Assay Buffer (PAB): 10 mM HEPES-NaOH, pH
8.0, 50 mM NaCl, 1 mM Na 2 EDTA, 250 mM sucrose. Store
solution at 4°C.
2. Proteasome Lysis Buffer (PLB): PAB with 0.2% Triton X-100
added fresh. Keep at 4°C.
3. Stock solutions of fluorogenic substrates: Commercially avail-
able fluorogenic substrates (e.g., Sigma-Aldrich, St. Louis,
MO) dissolved in dimethyl sulfoxide (DMSO) at 10 mM:
Suc-LLVY-amc, Boc-LSTR-amc and Z-LLE-amc. Store in
aliquots at −20°C and protect from light.
4. Stock solution of MG132: dissolved in DMSO at 20 mM.
Store in aliquots at −20°C.

2.2. Assay Solutions

Make the following assay solutions just before use in the 20S pro-
teasome activity assays (see Subheading 3.2 ):

1. Assay Buffer (AB): Add DMSO to PAB to a final concentra-
tion of 2% (20 ml of DMSO to each 1 ml of PAB).
2. Inhibitor Buffer (IB): For each 1 ml of PAB, add 20 ml of
stock solution of MG132 (20 mM in DMSO). Final concen-
tration of the inhibitor in the assay will be ~35 mM.
3. Substrate Solutions (protect from light):
1. Chymotrypsin-like activity : Add 20 ml of Suc-LLVY-amc
stock for each 1 ml of PAB. Final concentration of the sub-
strate in the assay will be ~21 mM.
2. Trypsin-like activity : Add 32 ml of Boc-LSTR-amc stock
for each 1 ml of PB. Final concentration of the substrate in
the assay will be ~34 mM.
3. Caspase-like activity : Add 100 ml of Z-LLE-amc stock for
each 1 ml of PB. Final concentration of the substrate in the
assay will be ~105 mM.

3. Methods

3.1. Sample
Preparation

For measuring the three proteasomal activities in triplicate and for
protein estimation, about 100-200 mg of tissue or 0.5-3 × 10 6
cells (depending on cell size) will be sufficient, following the pro-
cedures outlined below.