We used the fluorogenic substrates Bz-VGR-AMC and Suc-LLVY-AMC to measure trypsin-like and chymotrypsin-like activities. Specifically, we incubated 10 µl of brain homogenate with each substrate to probe in a total of 200 µl of assay buffer (25mM HEPES, pH 7.5, 0.5mM EDTA, 0.05% NP-40) using black 96-well plates. We obtained kinetic readings at 37°C every 1.5 minutes for 60 min (excitation 360 nm, emission 460 nm) using the Synergy HT multi-mode microplate reader with the Gen5 software (BioTek, Winooski, VT). Readings were normalized to total protein concentrations assayed via a Coomassie Protein Assay Kit (Bradford, Thermo Scientific, Waltham, MA) following the manufacturer’s instructions. The experimenter was blinded to the group allocation.
Coomassie protein assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
The Coomassie Protein Assay Kit is a colorimetric assay used to quantify the total protein concentration in a sample. It utilizes Coomassie Brilliant Blue G-250 dye, which binds to proteins, resulting in a color change that can be measured spectrophotometrically.
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Lab products found in correlation
Synergy ht, by Agilent Technologies (3 mentions)
3 nitrotyrosine elisa kit, by Abcam (3 mentions)
Ab6002, by Abcam (3 mentions)
Pierce mitochondria isolation kit, by Thermo Fisher Scientific (3 mentions)
Nitrocellulose blotting membrane, by GE Healthcare (2 mentions)
Pageruler prestained protein ladder, by Thermo Fisher Scientific (2 mentions)
Anti actin, by Merck Group (2 mentions)
Anti iκbα, by Santa Cruz Biotechnology (2 mentions)
Amersham ecl select western blotting detection reagent, by GE Healthcare (2 mentions)
Westran clear signal pvdf membranes, by Cytiva (2 mentions)
F chemibis 3, by DNR Bio-Imaging Systems (2 mentions)
Nupage novex 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (2 mentions)
Protran 0.2 mm nitrocellulose, by GE Healthcare (2 mentions)
Donkey anti rabbit hrp, by Stratech Scientific (2 mentions)
Donkey anti mouse hrp, by Stratech Scientific (2 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (2 mentions)
Luciferase assay system, by Promega (2 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
57 protocols using coomassie protein assay kit
1
Fluorogenic Proteasome Activity Assay
2
Quantitative Analysis of MET Levels
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Each tumor, untreated or treated, was sonicated for 30 seconds on ice and centrifuged at 12,000 rpm for 10 minutes. Supernatants were collected and protein levels were measured using the Coomassie Protein Assay Kit (Thermo Scientific, Rockford, IL). Protein levels were calculated from the standard curve obtained by protein standard, bovine serum albumin (BSA). MET levels were determined with an HPLC procedure described previously [29 (link), 58 (link)]. Standardized MET levels were calculated using the following formula: MET level (nmol/mg protein) = MET level (nmol/ml) / protein level (mg protein/ml) [7 (link)].
3
Optimized Refolding of Inclusion Bodies
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Purified inclusion bodies (ICs) were solubilized in 6 M of guanidinium-HCl. A set of refolding buffers was used in a stepwise solubilization strategy (ProteoStat kit, Enzo, Lörrach, Germany). Finally, an optimized refolding buffer was identified consisting of 50 mM of Tris, a pH of 8.3, 20 mM of NaCl, 0.8 mM of KCl, 0.8 M of L-Arginine, and 0.12 M of sucrose. Antigenic properties in solid phase ELISA were assayed using polyclonal antibodies raised in chickens or with specific monoclonal antibodies. The final protein concentration in refolding buffer was determined using a Coomassie protein assay kit (ThermoScientific, Rockford, IL, USA) and proteins were stored at 4 °C until further use.
4
Quantifying Urinary Protein Excretion
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Urinary protein excretion was measured using a Coomassie protein assay kit
(Thermo Fisher Scientific). Serum creatinine was measured using a Duppon ARL
analyser by Department of Biochemistry, Monash Health.
(Thermo Fisher Scientific). Serum creatinine was measured using a Duppon ARL
analyser by Department of Biochemistry, Monash Health.
5
Recombinant Protein Purification and Lipid Binding Assay
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Protein expression in E. coli BL21 (DE3) and subsequent purification were done as described previously [15 (link)]. The concentration of purified protein was measured using a Coomassie Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA). The purified protein was subjected to SDS-PAGE and visualized with Coomassie brilliant blue R-250 to check its purity.
Lipid overlay assays were conducted as recommended by the manufacture’s protocol. Briefly, the membrane (PIP Strips or Membrane Lipid Arrays; Echelon Bioscience Inc) was incubated in 3% fatty acid free BSA (Sigma-Aldrich) in a mixture of phosphate-buffered saline and 0.1% Tween 20 (PBST) for 1 h at room temperature (RT) and then incubated in the same solution containing 500 ng of purified recombinant protein for 1 h at RT. After washing three times with PBST, the membrane was incubated with a mouse anti-FLAG antibody (1:10000; Sigma-Aldrich) for 1 h at RT, followed by three washes with PBST. An anti-mouse IgG conjugated with horseradish peroxidase (1:10000; KPL) was used as a secondary antibody. Binding of proteins to phospholipids was visualized by incubation with a chemiluminescent substrate.
Lipid overlay assays were conducted as recommended by the manufacture’s protocol. Briefly, the membrane (PIP Strips or Membrane Lipid Arrays; Echelon Bioscience Inc) was incubated in 3% fatty acid free BSA (Sigma-Aldrich) in a mixture of phosphate-buffered saline and 0.1% Tween 20 (PBST) for 1 h at room temperature (RT) and then incubated in the same solution containing 500 ng of purified recombinant protein for 1 h at RT. After washing three times with PBST, the membrane was incubated with a mouse anti-FLAG antibody (1:10000; Sigma-Aldrich) for 1 h at RT, followed by three washes with PBST. An anti-mouse IgG conjugated with horseradish peroxidase (1:10000; KPL) was used as a secondary antibody. Binding of proteins to phospholipids was visualized by incubation with a chemiluminescent substrate.
6
Quantification of Tumor Methionine Levels
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Each tumor was sonicated for 30 seconds on ice and centrifuged at 12,000 rpm for 10 minutes. Supernatants were collected and protein levels were measured using the Coomassie Protein Assay Kit (Thermo Scientific, Rockford, IL). Protein levels were calculated from a standard curve obtained with a protein standard, bovine serum albumin (BSA). L-methionine levels were determined with the HPLC procedure described previously [47 (link), 64 (link)]. Standardized L-methionine levels were calculated per mg tumor protein.
7
Cell Signaling Transduction Profiling
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Cell lines profiled for signaling transduction analysis were seeded in technical replicates (n = 6) in 6-well plates and cultured until 80% confluent. Cells were washed twice with PBS (Invitrogen Life Technologies, Carlsbad, CA, USA) and lysed in Tissue Protein Extraction Reagent (T-PER) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 300 mM sodium chloride and a mixture of protease and phosphatase inhibitors to prevent protein degradation and preserve the integrity of the phosphoproteome, as previously described [19 (link)]. Protein concentration in each sample was assessed using the Coomassie Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) following manufacturer’s instructions. Lysates were first diluted to 1 µg/µL in T-PER and subsequently brought to a final concentration of 0.5 µg/µL in 2X Tris-Glycine SDS Sample buffer (Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 5% β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA). Lastly, lysates were boiled for 8 min at 100 °C and stored at −80 °C.
8
Fluorogenic Proteasome Activity Assay
9
Western Blot Protein Detection Protocol
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Cell lysates were prepared with lysis buffer [150 mM KCl, 75 mM Hepes of pH 7.5, 1.5 mM EGTA, 1.5 mM MgCl2, 10% glycerol, 0.1% NP-40, 30 mg/ml DNase, 30 mg/ml RNase, complete protease inhibitor cocktail (Roche), and complete phosphatase inhibitor cocktail (Sigma)]. Protein concentration of cell lysate was measured using the Coomassie protein assay kit (Thermo Scientific). Proteins were separated on SDS-PAGE, electroblotted onto a nitrocellulose blotting membrane (Amersham, GE Healthcare), and subjected to immunodetection using appropriate primary antibodies. Blocking and antibody incubations were performed in 5% nonfat dry milk. Proteins were visualized using horseradish peroxidase-conjugated secondary antibodies diluted at 1:2000 (Amersham) and the ECL system, according to the manufacturer’s instructions (Thermo Scientific).
10
Nucleic Acid Extraction and Validation
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