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.
Boc lstr amc
Manufactured by Merck Group
Sourced in United Kingdom, United States
Boc-LSTR-AMC is a fluorogenic substrate used for the detection and measurement of protease activity. It is composed of the amino acid sequence Boc-Leu-Ser-Thr-Arg, which is cleaved by proteases, releasing the fluorescent reporter molecule 7-amino-4-methylcoumarin (AMC). This substrate can be utilized in various biochemical assays to monitor the activity of proteases that recognize and cleave the Boc-LSTR peptide sequence.
Automatically generated - may contain errors
Lab products found in correlation
Mg132, by Merck Group (2 mentions)
Synergy 4 instrument, by Agilent Technologies (2 mentions)
Suc llvy amc, by Thermo Fisher Scientific (2 mentions)
Z lle amc, by Thermo Fisher Scientific (2 mentions)
Z lle amc, by Enzo Life Sciences (1 mentions)
Human 20s proteasome, by Enzo Life Sciences (1 mentions)
Suc llvy amc, by Enzo Life Sciences (1 mentions)
Mithras lb 940, by Berthold Technologies (1 mentions)
Z leu leu leu al mg132, by Merck Group (1 mentions)
Z lle amc, by Merck Group (1 mentions)
4 protocols using boc lstr amc
1
Proteasome Activity Assay in Liver
2
Proteasome Activity Assay with Brusatol
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The human 20S proteasome was obtained from Enzo Life Sciences, and 1 µg of purified protein was preincubated for 30 min at 37 °C with the indicated concentrations of brusatol or MG132 in 50 mM Hepes (pH 7.8). Probe substrates for trypsin-like (Boc-LSTR-AMC, 50 µM; Sigma–Aldrich), chymotrypsin-like (Suc-LLVY-AMC, 50 µM; Enzo Life Sciences, UK), or caspase-like (Z-LLE-AMC, 400 µM; Enzo Life Sciences) activity were subsequently added and substrate cleavage was determined by quantification of 7-amino-4-methylcoumarin (AMC) fluorescence (excitation 360 nm, emission 460 nm) over 8 h and referenced to an AMC standard curve.
3
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.
4
Proteasome Activity Assay in Cell Lysates
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Proteasome activities of 20S core: CT-L (chymotrypsin-like), T-L (trypsin-like), C-L (caspase-like) were assayed in the supernatants (20 µM) using specific fluorogenic substrate (70 µ M ) for each activity respectively Suc-LLVY-AMC (Calbiochem,USA), Boc-LSTR-AMC (Sigma Aldrich,France) and Z-LLE-AMC (Sigma Aldrich, France) and calculated as the difference between the presence or the absence of specific proteasome inhibitor MG132 (Z-Leu-Leu-Leu-al (Sigma Aldrich,France) (10 µM) for 1 h at 37 °C as previously described39 (link). Activities were expressed as relative fluorescent units (RFU)/60 min/mg proteins with (355 excitation/460 emission) by microtiter plate fluorometer Mithras LB940 (Berthold Technologies, Bad Wilbad, Germany) .
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