Determination of Proteasomal Activities - Protein Misfolding and Cellular Stress in Disease and Aging - page 183

should be higher than 1 mg/ml, otherwise decrease the added
PLB to the samples accordingly.
5. If a microplate reader fluorometer is not available, a kinetic
assay can be replaced by end point measurements. For that,
make the assay as mentioned in the Subheading 3.2 in micro-
tubes at 37°C (protecting from light) and stop the reaction
with 300 ml of an acidic solution (30 mM sodium acetate,
70 mM acetic acid, 100 mM sodium chloroacetate, pH 4.3).
After addition of 2 ml of distilled water, read the fluorescence
in a fluorometer with the proper excitation and emission fil-
ters. The time of incubation has to be previously tested and
has to be within the linear response.
6. To calibrate the plate reader fluorometer, a calibration curve
with a range of AMC concentrations (0.1-v10 mM) has to
give a linear response.
7. The quantity of protein necessary for accurate results for each
peptidase activity should be determined previously for each
cell type or organ/tissue with preliminary assays. The correct
protein load should give a linear response along the time (at
least in the first 30 min) and not exceed during the assay 1.5×
the signal generated by 5 mM of AMC standard. In addition,
to increase accuracy, the chosen protein quantity should be
pipetted in a similar volume between samples, that is, different
samples should have similar protein concentration, preferably
higher than 1 mg/ml. If the pippetted volume of sample dif-
fers much between samples, since the fluorescence intensity
also depends on the height of the liquid in a well, the compari-
son of the results between different samples may be affected.
8. Pipet also a standard curve, or at least one standard within the
linear range (e.g., 5 mM AMC), if the results are going to be
given in nmol of AMC produced/min/mg protein (see
Subheading 3.3 ). For the standards, one should take the fluo-
rescent intensity in an endpoint measurement, and not the
slope in a kinetic assay as in the samples.
9. This pre-incubation procedure will allow AB and IB to stabi-
lize at 37°C and also to give time to the MG132 to com-
pletely inhibit proteasome.

References

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2. Shringarpure R, Grune T, Davies KJA (2001)
Protein oxidation and 20S proteasome-
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3. Jung T, Grune T (2008) The proteasome and
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4. Coux O, Tanaka K, Goldberg A (1996)
Structure and functions of the 20S and 26S
proteasomes. Annu Rev Biochem 65:801-807