In vitro 26S proteasome activity analyses were carried out as previously described [61] (link). Briefly, using a Precellys 24 homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France), animals were lysed using proteasome activity assay buffer containing 50 mM Tris-HCl (Ph 7.5), 250 mM sucrose, 5 mM MgCl2, 2 mM ATP, 1 mM dithiothreitol and 0.5 mM EDTA. The lysate was centrifuged at 10,000 g for 15 min at 4°C. For each assay, 25 µg of total lysate was loaded into each well of a 96-well microtiter plate, after which fluorogenic substrate was added. For determining the chymotrypsin-like activity of the proteasome, Z-Gly-Gly-Leu-AMC (Enzo Life Sciences, Farmingdale, NY) was used as a substrate. After incubation for 1 h at 25°C, fluorescence (an excitation wavelength 380 nm and an emission wavelength 460 nm) was measured with a SpectraMax M2 Microplate Reader (Molecular Devices, Silicon Valley, CA).
Z gly gly leu amc
Manufactured by Enzo Life Sciences
Sourced in United States
Z-Gly-Gly-Leu-AMC is a fluorogenic peptide substrate. It is used for the detection and quantification of protease activity in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Spectramax m2 microplate reader, by Molecular Devices (3 mentions)
Precellys 24, by Bertin Technologies (2 mentions)
96 well microtiter plate, by BD (2 mentions)
Enspire, by PerkinElmer (2 mentions)
Precellys 24 homogenizer, by Bertin Technologies (1 mentions)
Suc leu leu val tyr amc, by Enzo Life Sciences (1 mentions)
Z leu leu glu amc, by Enzo Life Sciences (1 mentions)
Ac arg leu arg amc, by Enzo Life Sciences (1 mentions)
5 protocols using z gly gly leu amc
1
Proteasome Activity Assay Protocol
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In vitro 26S proteasome activity analyses were carried out as previously described [61] (link). Briefly, using a Precellys 24 homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France), animals were lysed using proteasome activity assay buffer containing 50 mM Tris-HCl (Ph 7.5), 250 mM sucrose, 5 mM MgCl2, 2 mM ATP, 1 mM dithiothreitol and 0.5 mM EDTA. The lysate was centrifuged at 10,000 g for 15 min at 4°C. For each assay, 25 µg of total lysate was loaded into each well of a 96-well microtiter plate, after which fluorogenic substrate was added. For determining the chymotrypsin-like activity of the proteasome, Z-Gly-Gly-Leu-AMC (Enzo Life Sciences, Farmingdale, NY) was used as a substrate. After incubation for 1 h at 25°C, fluorescence (an excitation wavelength 380 nm and an emission wavelength 460 nm) was measured with a SpectraMax M2 Microplate Reader (Molecular Devices, Silicon Valley, CA).
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In vitro 26S proteasome activity analyses were carried out as previously described [61] (link). Briefly, using a Precellys 24 homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France), animals were lysed using proteasome activity assay buffer containing 50 mM Tris-HCl (Ph 7.5), 250 mM sucrose, 5 mM MgCl2, 2 mM ATP, 1 mM dithiothreitol and 0.5 mM EDTA. The lysate was centrifuged at 10,000 g for 15 min at 4°C. For each assay, 25 µg of total lysate was loaded into each well of a 96-well microtiter plate, after which fluorogenic substrate was added. For determining the chymotrypsin-like activity of the proteasome, Z-Gly-Gly-Leu-AMC (Enzo Life Sciences, Farmingdale, NY) was used as a substrate. After incubation for 1 h at 25°C, fluorescence (an excitation wavelength 380 nm and an emission wavelength 460 nm) was measured with a SpectraMax M2 Microplate Reader (Molecular Devices, Silicon Valley, CA).
2
Proteasome Activity Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Briefly, using a Precellys 24 homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France), animals were lysed in a proteasome activity assay buffer containing 50 mM Tris-HCl (pH 7.5), 250 mM sucrose, 5 mM MgCl2, 2 mM ATP, 1 mM dithiothreitol and 0.5 mM EDTA. The lysate was centrifuged at 10,000 g for 15 min at 4°C. For each experiment, 25 μg of total lysate was loaded into each well of a 96-well microtiter plate, after which a fluorogenic substrate was added. For testing the chymotrypsinlike activity of the proteasome, Z-Gly-Gly-Leu-AMC (Enzo Life Sciences, Farmingdale, NY) was used as a substrate. After incubation for 1 h at 25°C, fluorescence (an excitation wavelength of 380 nm and an emission wavelength of 460 nm) was detected with a SpectraMax M2 Microplate Reader (Molecular Devices, Silicon Valley, CA).
3
Proteasome Activity Assay in C. elegans
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Our proteasome activity assays in C. elegans used a previously described method, with slight modifications.64 (link) Briefly, using a Precellys 24 homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France), worms were lysed in a proteasome activity assay buffer containing 50 mM Tris-HCl (pH 7.5), 250 mM sucrose, 2 mM adenosine triphosphate (ATP), 5 mM MgCl2, 1 mM dithiothreitol, and 0.5 mM ethylenediaminetetraacetic acid (EDTA). The lysate was centrifuged at 10,000g for 15 min at 4 °C. For each test, 25 μg of total lysate was loaded into each well of a 96-well microtiter plate, after which fluorogenic substrate was added. Z-Gly-Gly-Leu-AMC (Enzo Life Sciences, Farmingdale, NY, USA) was used as a substrate for testing the chymotrypsin-like activity of the proteasome. After incubation for 1 h at 25 °C, fluorescence (excitation wavelength = 380 nm, emission wavelength = 460 nm) was measured with a SpectraMax M2 Microplate Reader (Molecular Devices, Silicon Valley, CA, USA).
4
Assay of 26S Proteasome Activity
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The in vitro assay of 26 S proteasome activities was performed as previously described136 (link). Cells were collected in proteasome activity assay buffer (50 mM Tris-HCl, pH7.5, 250 mM sucrose, 5 mM MgCl2, 0.5 mM EDTA, 2 mM ATP and 1 mM DTT) and lysed by passing ten times through syringe needle (27 G). Then, lysate was centrifuged at 10,000 g for 10 min at 4 °C. 25 μg of total protein of cell lysates were transferred to a 96-well microtiter plate (BD Falcon) and incubated with the fluorogenic substrate Z-Gly-Gly-Leu-AMC (Enzo Lifescience). Fluorescence (380 nm excitation, 460 nm emission) was monitored on a microplate fluorometer (EnSpire, Perkin Elmer) every 5 min for 2 h at 37 °C.
5
Proteasome Activity Assay Protocol
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Cells were collected in proteasome activity assay buffer (50 mM Tris-HCl, pH 7.5, 250 mM sucrose, 5 mM MgCl2, 0.5 mM EDTA, 2 mM ATP and 1 mM DTT) and lysed by passing 10 times through a 27 G needle attached to a 1 ml syringe needle. Lysates were centrifuged at 10,000 × g for 10 min at 4 °C. Then, 25 μg of total protein of cell lysates were transferred to a 96-well microtiter plate (BD Falcon) and incubated with the fluorogenic proteasome substrate. To measure the chymotrypsin-like activity of the proteasome we used either Z-Gly-Gly-Leu-AMC (Enzo) or Suc-Leu-Leu-Val-Tyr-AMC (Enzo). We used Z-Leu-Leu-Glu-AMC (Enzo) to measure the caspase-like activity of the proteasome, and Ac-Arg-Leu-Arg-AMC (Enzo) for the proteasome trypsin-like activity. Fluorescence (380 nm excitation, 460 nm emission) was monitored on a microplate fluorometer (EnSpire, Perkin Elmer) every 5 min for 1 h at 37 °C.
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