20S proteasomal activity was quantified in plasma fraction by monitoring the accumulation of the fluorescent cleavage product 7‐amino‐4‐methylcoumarin (AMC) from a synthetic proteasomal substrate.28 The fluoropeptide Suc‐Leu‐Leu‐Val‐Tyr‐AMC (Sigma‐Aldrich) was used to measure the chymotrypsin‐like activity of the proteasome.
Suc leu leu val tyr amc
Manufactured by Merck Group
Sourced in United States
Suc-Leu-Leu-Val-Tyr-AMC is a fluorogenic substrate used for the detection and measurement of protease activity. It consists of a pentapeptide sequence (Suc-Leu-Leu-Val-Tyr) coupled to the fluorescent reporter molecule 7-amino-4-methylcoumarin (AMC). Upon cleavage by a protease, the AMC moiety is released, resulting in an increase in fluorescent signal that can be quantified.
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Lab products found in correlation
Spectramax m2 microplate reader, by Molecular Devices (3 mentions)
Boc leu ser thr arg amc, by Merck Group (3 mentions)
Ls 30 spectrofluorometer, by PerkinElmer (2 mentions)
Ac gly pro leu asp amc, by Merck Group (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (2 mentions)
Enspire multimode plate reader, by PerkinElmer (1 mentions)
Enspire multimode reader, by PerkinElmer (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Leupeptin, by Merck Group (1 mentions)
Mg132, by Selleck Chemicals (1 mentions)
Microplate reader, by Molecular Devices (1 mentions)
Suc leu leu val tyr amc, by Bachem (1 mentions)
Mg132, by Merck Group (1 mentions)
Aprotinin, by Merck Group (1 mentions)
Z leu ser thr arg amc, by Merck Group (1 mentions)
Z leu leu glu amc, by Merck Group (1 mentions)
Z gly pro ala leu ala mca, by Biomatik (1 mentions)
Ecl system, by Cytiva (1 mentions)
14 protocols using suc leu leu val tyr amc
1
Quantifying 20S Proteasomal Activity
2
Proteasome Activity Assay in Worms
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We used the previously described method to determine the proteasome activity (chymotrypsinlike activity) in worms [31 (link)]. Worms were first lysed by using a Precellys 24 homogenizer and proteasome activity assay buffer [50 mM Tris-HCl (pH = 7.5), 250 mM sucrose, 2 mM ATP, 5 mM MgCl2, 1 mM dithiothreitol, and 0.5 mM EDTA]. The lysate was then centrifuged at 10,000× g at 4 °C for 15 min. For each sample, 25 μg of total lysate was added to each well of a 96-well microtiter plate, and then the fluorescent substrate Suc-Leu-Leu-Val-Tyr-AMC (Sigma-Aldrich, St. Louis, MO, USA) was added. After the plates were incubated for 1 h at 25 °C, the fluorescence was measured with a SpectraMax M2 Microplate Reader (Molecular Devices, Silicon Valley, CA, USA) (λex = 380; λem = 460 nm).
3
Proteasome Activity Assay
4
Quantifying Proteasome Chymotrypsin-like Activity
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After drug treatments, proteasome activity was analyzed in cell lysates in lysis buffer containing 8.56 g sucrose, 0.6 g HEPES, 0.2 g MgCl2, 0.037 g EDTA in 100 mL H2O. Following three freeze-thaw cycles the lysates were centrifuged at 14000 g for 30 min and the supernatant was used to measure the proteasome activity. To measure chymotrypsin-like activity of the proteasome the fluorogenic peptide substrate (Suc-Leu-Leu-Val-Tyr-AMC, Sigma-Aldrich) was used at a concentration of 200 μM in a reaction mixture containing 225 mM Tris buffer (pH 7.8), 7.5 mM MgOAc, 7.5 mM MgCl2, 45 mM KCl, and 1 mM dithiothreitol. ATP was added to the reaction mixture to stimulate 26S proteasome activity. 20S proteasome activity is distinguished by depleting ATP with addition of deoxy-glucose and hexokinase in the reaction mixture. The samples were incubated at 37 °C for 30 min, and the fluorescence of free methyl coumarin (MCA) was measured using an Enspire multimode plate reader at 360 nm excitation/485 nm emission (PerkinElmer). Free MCA was used as the standard for quantification. Results were calculated according to the standard curve that was prepared by using free MCA.
5
Proteasome Activity and AVO Staining
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For the proteasome activity assay (chymotrypsin-like activity) and acidic vesicular organelle (AVO) staining (acridine orange, 0.5 μg/mL) in the SH-SY5Y cell line, we followed the experimental method described in the previous study [9 (link),10 (link)]. The chymotrypsin-like activity of the proteasome was detected in cell lysates with Suc-Leu-Leu-Val-Tyr-AMC (Sigma-Aldrich, St. Louis, MO, USA) as the substrate. After incubation for 1 h at 25 °C, fluorescence was measured with a SpectraMax M2 Microplate Reader (Molecular Devices, Silicon Valley). In the AVO staining, cells were washed with PBS and incubated with acridine orange hydrochloride solution for 10 min at 37 °C in the dark. The formation of AVO was detected under the fluorescence microscope. The images were quantified for fluorescence intensity using ImageJ software (National Institutes of Health).
6
Proteasome Activity Assay in Worms
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Proteasome activity (chymotrypsin-like activity) was measured in worms using a previously described method [76 (link)]. Briefly, using a Precellys 24 homogenizer, 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 10,000× g 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. Suc-Leu-Leu-Val-Tyr-AMC (Sigma-Aldrich, St. Louis, MO, 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).
7
Proteasome Activity Quantification Protocol
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Proteasome β5 subunit activity was directly measured with the hydrolysis of the fluorogenic substrate Suc-Leu-Leu-Val-Tyr-AMC (Sigma-Aldrich). The MCF-7 cells were treated with 5, 10, and 25 μM BrPQ5 and 25 μM DOXO along with control for 24 h. Then, the cells were scraped on ice, and cell lysates were prepared in lysis buffer (0.25 M saccharose, 25 mM HEPES, 10 mM MgCl2, 1 mM EDTA, 1 mM DTT) with three freeze–thaw cycles. The lysates were centrifuged (13,500 rpm, 30 min, 4 °C). The total protein concentration was determined using the Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Free AMC concentration was used to create a standard curve for quantitative analysis. The proteasome activity was measured from the supernatants after 30 min incubation with the fluorogenic substrate in buffer containing 225 mM Tris, 45 mM potassium chloride, 7.5 mM magnesium acetate, 7.5 mM magnesium chloride, and 1 mM DTT, pH 7.8 using EnSpire multimode reader (PerkinElmer) at 360 nm excitation/460 nm emission.
8
Chymotrypsin-like Proteasome Activity Assay
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Liver and heart tissue were homogenized in a lysis buffer containing 100 mM Tris-HCl, 145 mM NaCl, 10 mM EDTA, and 0.5% Triton X-100 at pH 7.5 as previously described by us [27 (link)]. Chymotrypsin-like activity was evaluated by fluorescence in a reaction buffer consisting of 50 mM Tris, 20 mM KCl, 1 mM magnesium acetate, 2 mM dithiothreitol, 1 mM leupeptin (Sigma-Aldrich), 1 mM phenylmethylsulfonyl fluoride (PMSF; Merck) and Suc-Leu-Leu-Val-Tyr-AMC (Sigma-Aldrich; 50 μM) as a substrate. Proteasome activity was fluorimetrically measured at λexc. 355 nm and at λem. 460 nm and is given in arbitrary fluorescence units per mg protein. Protein concentrations were determined using the Bradford assay.
9
Calpain Activity Assay Using Fluorogenic Substrate
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The polypeptide Suc-Leu-Leu-Val-Tyr-AMC (Sigma-Aldrich) was used to assess calpain activity [31 (link)]. Briefly, 40 μL of trophozoites protein extracts (1 mg protein/mL) were added to 160 μL of 50 μM Suc-Leu-Leu-Val-Tyr-AMC in 100 mM Tris/HCl, 145 mM NaCl, pH 7.3. Then, calpain activity was assayed with 10 mM Ca2+ 7-amino-4-methylcoumarin (AMC) (Sigma-Aldrich). Released AMC was measured by fluorescence spectroscopy using 360 nm excitation and 430 nm emission filters. Standard curves were generated for each experiment using AMC of known concentrations (0–25 μM). Calpain activity was expressed as pmol of AMC released per mg of total protein at 10 mM Ca2+.
10
Proteasome Activity Assay Protocol
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Cells were lysed using lysis buffer containing 25 mmol/L Tris-HCl, pH 7.5; 100 mmol/L KCl; 1 mmol/L EDTA; 0.1% (v/v) Triton X-100; and 1 mmol/L phenylmethane sulfonylfluoride. Cell lysates (10 μg protein/each) were resuspended in 25 mmol/L Tris-HCl, pH 7.4; 1 mmol/L dithiothreitol; and 20 mmol/L KCl. The proteasome activities were measured with 100 μmol/L each of fluorogenic substrates: Z-Leu-Leu-Glu-7-amino-4- methylcoumarin (AMC) for caspase-like, Suc-Leu-Leu-Val-Tyr-AMC for chymotrypsin-like, and Boc-Leu-Ser-Thr-Arg-AMC (Sigma-Aldrich Saint Louis, MO, USA) for trypsin-like activity in the absence or presence of 25 μmol/L proteasome inhibitor MG-132 (Selleckchem, S2619, Houston, TX, USA). The change in fluorescence was monitored for 2 h at 37 °C in a microplate fluorescence reader with an excitation wavelength of 380 nm and emission wavelength of 460 nm. The activity was calculated after subtracting the background (part not inhibited by MG-132).
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