For measuring proteasome activity in vitro from liver extracts we utilized fluorogenic model substrates as described (Cui et al., 2014 (link)). Briefly, liver was Dounce homogenized in proteasome reaction buffer (50 mM Tris, 1mM EDTA, 150 mM NaCl, 5 mM MgCl2, 0.5 mM DTT, pH 7.5) and clarified by centrifugation. 20 μg of liver protein was then incubated with 100 μM of Suc-LLVY-AMC (Fisher Scientific), Z-LLE-AMC (Fisher Scientific), or Boc-LSTR-AMC (Sigma) to measure chymotrypsin, caspase, and trypsin-like proteasomal activities, respectively, plus freshly added 0.1 mM ATP and 0.5 mM DTT. As a negative control duplicate samples were incubated with mg132 (Sigma) as described (Cui et al., 2014 (link)). The fluorescence accumulation was monitored for 2 hours at 37°C and an excitation/emission of 380/460 using a BioTek Synergy 4 instrument. Fluorescence was converted to nmol of AMC cleaved per minute per mL and normalized by dividing individual activities by the average assay activity.
Z lle amc
Manufactured by Thermo Fisher Scientific
Z-LLE-AMC is a fluorogenic substrate used for the detection and quantification of caspase-3 and caspase-7 activity in cell-based and cell-free assays. The substrate consists of the amino acid sequence Z-Leu-Leu-Glu linked to the fluorogenic reporter molecule 7-amino-4-methylcoumarin (AMC). Upon cleavage by caspase-3 or caspase-7, AMC is released, resulting in an increase in fluorescent signal that can be measured.
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2 protocols using z lle amc
1
Proteasome Activity Assay in Liver
2
Proteasome Activity Assay in Liver
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For measuring proteasome activity in vitro from liver extracts we utilized fluorogenic model substrates as described (Cui et al., 2014 (link)). Briefly, liver was Dounce homogenized in proteasome reaction buffer (50 mM Tris, 1mM EDTA, 150 mM NaCl, 5 mM MgCl2, 0.5 mM DTT, pH 7.5) and clarified by centrifugation. 20 μg of liver protein was then incubated with 100 μM of Suc-LLVY-AMC (Fisher Scientific), Z-LLE-AMC (Fisher Scientific), or Boc-LSTR-AMC (Sigma) to measure chymotrypsin, caspase, and trypsin-like proteasomal activities, respectively, plus freshly added 0.1 mM ATP and 0.5 mM DTT. As a negative control duplicate samples were incubated with mg132 (Sigma) as described (Cui et al., 2014 (link)). The fluorescence accumulation was monitored for 2 hours at 37°C and an excitation/emission of 380/460 using a BioTek Synergy 4 instrument. Fluorescence was converted to nmol of AMC cleaved per minute per mL and normalized by dividing individual activities by the average assay activity.
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