Determination of Proteasomal Activities - Protein Misfolding and Cellular Stress in Disease and Aging - page 182

3. Calculate proteasome specific activity using previous equation:
Proteasome specific activity = (20 × 5 × 0.23)/(300 × 0.01) = 7.67 nmol
AMC/min/mg
4. Conclusions
With the present protocol, the three different activities of the
proteasome can be estimated in cell lysates using fluorogenic sub-
strates. By measuring the 20S proteasomal activity in cell homo-
genates, many sources of interferences may occur, such as due to
the use of the general proteasome inhibitor MG132 that may also
inhibit other proteases, or due to the lack of 100% specificity of
the proteasome substrates. However, if the protocol is performed
as described above and by using the correct experiment controls
run in parallel with the treatment conditions, it will give effective
estimation of any changes in proteasomal activity. In addition, in
opposition to the end-point measurements, estimation of protea-
somal activities in a microplate reader fluorometer by following
the kinetics of the reaction along the time will increase the accu-
racy of the assay, if care is taken to pipette precise volumes of the
different solutions and protein.
Finally, it may be noted that the above protocols for the esti-
mation of proteasomal activities have been successfully applied for
determining changes during aging, anti-aging interventions, cell
cycle analysis, and in various disease states, including neurode-
generative diseases and cancers ( 15-18 ).
5. Notes
1. Clean supernatant can be stored in aliquots at −80°C (or
−20°C for shorter time) before proteasome activity assays,
but it will decrease the activity by about 10% per each thaw-
ing. Avoid repeated freeze/thaw cycles of the sample.
2. As a guide, use 300 ml of PLB for each T75 flask or 60 ml per
well of a 6-well plate. The final concentration of protein of
the homogenate should be higher than 1 mg/ml, otherwise
decrease the added PLB to the samples accordingly.
3. Cell pellets can be then either immediately used for prepara-
tion of cell homogenates, or stored at −80°C for later homog-
enization and proteasome activities assays.
4. As a guide, use 250 ml of PLB for a cell pellet taken from a T75
flask. The final concentration of protein of the homogenate