One μg of total protein from sample homogenates was used. The reaction mixture contained 50 mM Tris–HCl pH = 8, 1 mM DTT, and 40 μM Suc-Leu-Leu-Val-Tyr-AMC (Sigma Aldrich) substrate for chymotrypsin-like activity or 40 μM of Boc-Leu-Ser-Thr-Arg-AMC for trypsin-like activity or 100 μM of Ac-Gly-Pro-Leu-Asp-AMC (Sigma Aldrich) for Caspase-like activity. To distinguish the 26S proteasome activity from the 20S proteasome activity, 5 mM ATP was added to the reaction mixture. The mixture was then incubated for 30 min at 37 °C. The reaction was stopped by adding 100 mM monochloroacetate and 30 mM sodium acetate pH = 4.3. Fluorescence was determined by measuring the release of AMC (λ excitation: 370 nm, λ emission: 410 nm), using a Perkin Elmer LS 30 spectrofluorometer.
Boc leu ser thr arg amc
Manufactured by Merck Group
Sourced in United States, Germany, Switzerland
Boc-Leu-Ser-Thr-Arg-AMC is a synthetic peptide substrate used for the fluorometric detection and measurement of protease activity. It consists of the amino acid sequence Boc-Leu-Ser-Thr-Arg, which is linked to the fluorogenic reporter molecule 7-amino-4-methylcoumarin (AMC). Upon cleavage by proteases, the AMC is released, resulting in an increase in fluorescence that can be detected and quantified.
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Lab products found in correlation
Suc leu leu val tyr amc, by Merck Group (3 mentions)
Ls 30 spectrofluorometer, by PerkinElmer (2 mentions)
Ac gly pro leu asp amc, by Merck Group (2 mentions)
N succinyl leu leu val tyr amc, by Merck Group (2 mentions)
Mg132, by Selleck Chemicals (1 mentions)
Z phe arg amc, by Bachem (1 mentions)
Z arg arg amc, by Merck Group (1 mentions)
Milli q system, by Merck Group (1 mentions)
5 protocols using boc leu ser thr arg amc
1
Proteasome Activity Assay
2
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).
3
Proteasome Activity Assay
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One μg of total protein from sample homogenates was used. The reaction mixture contained 50 mM Tris–HCl pH = 8, 1 mM DTT, and 40 μM Suc-Leu-Leu-Val-Tyr-AMC (Sigma Aldrich) substrate for chymotrypsin-like activity or 40 μM of Boc-Leu-Ser-Thr-Arg-AMC for trypsin-like activity or 100 μM of Ac-Gly-Pro-Leu-Asp-AMC (Sigma Aldrich) for Caspase-like activity. 5 mM ATP was added to the reaction mixture to distinguish 26S proteasome activity from 20S proteasome activity. The mixture was then incubated for 30 min at 37 °C, and stopped by adding 100 μM monochloroacetate and 30 mM sodium acetate pH = 4.3. Fluorescence was determined by measuring the release of AMC (λ excitation: 370 nm, λ emission: 410 nm), using a Perkin Elmer LS 30 spectrofluorometer.
4
Characterization of Hsp Proteins
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Red was from JRH Biosciences. L-[3-14 C-alanine] dopa and L-[U-14 C] leucine were ! from Amersham Biosciences (GE Healthcare). N-succinyl-Leu-Leu-Val-Tyr-AMC (where Suc is succinyl and AMC is 7-amino-4-methylcoumarin) and Boc-Leu-Ser-Thr-Arg-AMC (where Boc is t-butoxycarbonyl) were purchased from Sigma Chemical Co. Z-Arg-Arg-AMC (where Z is benzyloxycarbonyl), Z-Phe-Arg-AMC and Ac-Nle-Pro-Nle-Asp-AMC (where Ac is acetyl and Nle is norleucine) were from Bachem AG. Anti-Hsp70 (cat # 386013) and anti-Hsp27 rabbit polyclonal (cat # 386035) were purchased from Calbiochem, Darmstadt, Germany. Anti-Hsp90 rabbit polyclonal (cat# ab53110) and HRP-conjugated anti-rabbit IgG H&L goat polyclonal secondary antibody (cat# ab6721) was purchased from Abcam Inc, Cambridge, UK.
All aqueous solutions and buffers were prepared using water filtered through a four-stage Milli-Q system (Millipore). All other chemicals, solvents and chromatographic materials were of analytical reagent or cell-culture grade.
All aqueous solutions and buffers were prepared using water filtered through a four-stage Milli-Q system (Millipore). All other chemicals, solvents and chromatographic materials were of analytical reagent or cell-culture grade.
5
Proteasome Activity Assay
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Proteasome chymotryptic, peptidylglutamyl-peptide-hydrolysing (PGPH) and tryptic activities were measured by the initial linear rates of cleavage of the fluorescent reporter group (7-amino-4-methylcoumarin/AMC) from peptide substrates N-Succinyl-Leu-Leu-Val-Tyr-AMC (Sigma-Aldrich Co., MO, USA), Acetyl-Nle-Pro-N [27] le-Asp-AMC (where Nle is norleucine; Bachem Holding AG, Bubendorf, Switzerland), Boc-Leu-Ser-Thr-Arg-AMC (where Boc is t-butoxycarbonyl; Sigma-Aldrich Co.) respectively (previously described in [22, 27] . Pelleted cells were resuspended in homogenising buffer containing 250 mM sucrose, 5 mM MgCl 2 , 2 mM ATP, 1 mM DTT, 0.025% digitonin, 0.5 mM EDTA and 50 mM Tris-HCl, pH 7.5 at 4°C. This homogenate was incubated on ice for 5 min to allow digitonin-mediated permeabilisation of the cell membrane followed by centrifugation at 20,000 x g for 15 min at 4°C. Samples were incubated for 30 min at room temperature in the presence or absence of the proteasome inhibitor epoxomicin (20 µM). Proteasome activity was measured in reaction buffer (0.05 mg/mL BSA, 40 mM KCl, 5 mM MgCl 2 , 0.5 mM ATP, 1 mM DTT and 50 mM Tris-HCl, pH7.5) with 100 µM chymotryptic/PGPH or 600 µM for the tryptic substrates by measuring the change in fluorescence (λ ex 360 nm and λ em 469 nm) for 30 min.
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