Frozen gastrocnemius samples (40–50 mg) were homogenized in 10× volume of ice‐cold buffer containing 30 mmol/L NaHepes (pH 7.4), 5 mmol/L EGTA, 3 mmol/L EDTA, 32% glycerol, 20 mmol/L KCl, HALT protease inhibitor cocktail (Thermo Fisher, Waltham, MA) and phosphate inhibitor cocktail 3 (Sigma‐Aldrich, St. Louis, MO). Tissue homogenates were centrifuged at 12,000g for 15 min at 4°C, and supernatants were collected. Protein concentration was assessed by the bicinchoninic acid method (BCA; Pierce Biotechnology, Inc., Rockford, IL), with BSA for the standard curve.
Phosphate inhibitor cocktail 3
Manufactured by Merck Group
Sourced in United States, Denmark
The Phosphate inhibitor cocktail 3 is a laboratory reagent designed to inhibit the activity of phosphatases in biological samples. It is a mixture of chemical compounds that work together to prevent the dephosphorylation of proteins and other biomolecules, allowing for the preservation of their phosphorylation state during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Phosphate inhibitor cocktail 2, by Merck Group (2 mentions)
Bicinchoninic acid method, by Thermo Fisher Scientific (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Carboxymethylcellulose, by Merck Group (1 mentions)
Phenol red, by Merck Group (1 mentions)
Trichloroacetic acid, by Merck Group (1 mentions)
Thiobarbituric acid, by Merck Group (1 mentions)
Evans blue, by Merck Group (1 mentions)
Buffered formalin, by Merck Group (1 mentions)
Leupeptin, by Merck Group (1 mentions)
Aprotinin, by Merck Group (1 mentions)
Serotonin, by Merck Group (1 mentions)
N acetylcysteine, by Merck Group (1 mentions)
N butanol, by Merck Group (1 mentions)
Acetylcholine, by Merck Group (1 mentions)
Malonaldehyde, by Merck Group (1 mentions)
Ferrous sulfate, by Merck Group (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Claudin 1, by Santa Cruz Biotechnology (1 mentions)
Loperamide, by Merck Group (1 mentions)
6 protocols using phosphate inhibitor cocktail 3
1
Gastrocnemius Muscle Protein Extraction
2
Colonic Contractility Evaluation Protocol
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Aqueous extracts HJE and PEP were generously provided by Professor Ji Hoon Jeong (Department of Pharmacology, College of Medicine, Chung-Ang University) on June 25, 2019. Tissues for colonic contractility were maintained in Krebs buffer [NaCl, 116.6 mM; NaHCO3, 21.9 mM; NaH2PO4, 1.2 mM; KCl, 3.4 mM; MgCl2, 1.2 mM; glucose, 5.4 mM; and CaCl2, 2.5 mM]. OCLN, claudin-1, and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Loperamide, Evans blue, 10% buffered formalin, reagent alcohol, N-acetyl cysteine, carboxymethylcellulose, phenol red, trichloroacetic acid, acetylcholine, serotonin, aprotinin, leupeptin, phosphate inhibitor cocktail-3, thiobarbituric acid, n-butanol, malonaldehyde, tripyridyl triazine (TPTZ), and ferrous sulfate were purchased from Sigma (St. Louis, MO, USA).
3
Hypergravity-Induced Protein Extraction and Analysis
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Immediately after loading hypergravity, the cells were lyzed in RIPA buffer containing 0.5% phosphate inhibitor cocktail 3 (Sigma), 1mM EDTA, 1mM NaF, and 2mM Na3VO4. The lysed cells were then centrifuged at 12,000 rpm for 5 min at 4°C, and the supernatants were mixed with loading buffer 2% SDS, 1M DTT, and Laemmli sample buffer (Bio-Rad)). The total protein quantification was carried out using the Bio-Rad DC protein assay kit (Bio-Rad). Based on the results of the protein quantification, equal amounts of protein in each sample were separated in a polyacrylamide gel (10% Acrylamide, 390mM Tris HCl (pH8.8), 0.05% SDS, milli-Q water, 0.04% TEMED, and 0.1% APS) by SDS-PAGE and transferred to PVDF membranes (Bio-Rad). After blocking the membranes with 5% non-fat milk in Tris Buffered Saline with Tween-20 (TBST) for 1 h at room temperature, the membranes were incubated with the primary antibody for 1 h at room temperature or overnight at 4°C. For visualizing the primary antibody, the membranes were incubated with an HRP-conjugated goat anti-rabbit IgG antibody in TBST for 1 h at room temperature. Protein bands were revealed by enhanced chemiluminescence (ECL, Roche) and imaged using a LAS-3000 imaging system (Fujifilm).
4
Purification and Interaction of GST Fusion Proteins
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GST proteins (GST only, GST‐Synr, GST‐Synr ΔBH3, and GST‐Synr Δccd) were expressed in BL21 E. coli (Takara #9126) and purified by affinity for glutathione Sepharose (GE healthcare). After preculture for 4 h, we applied a cold shock according to the manufacturer's instruction and incubated in LB media with 0.1 mM IPTG at 15°C for 20 h. Cell pellets were isolated by centrifugation and lysed by sonication in the E. coli lysis buffer (PBS, 1% NP40, 250 μM PMSF). GST proteins were purified by affinity for glutathione Sepharose. After washing beads, lysates that contain Flag‐tagged proteins or HA‐tagged proteins were added. To make the lysates, HEK293T cells were lysed in the cell lysis buffer (pH 7.4 50 mM Hepes, 2% NP40, 150 mM NaCl, 5% Glycerol, 1 mM MgCl2, 1 mM MnCl2, 10 mM NaF, 1 mM Na3VO4, 10 μg/ml Leupeptin, 2 μg/ml Pepstatin, 0.1% Aprotinin, 1% Protease inhibitor cocktail (Roche, cOmplete, EDTA free), 0.1% Phosphatase inhibitor cocktail 2 (P5726, Sigma), 0.1% Phosphate inhibitor cocktail 3 (P0044, Sigma), 250 μM PMSF and 1 mM DTT), and cell lysates were cleaned by centrifugation. After washing the GST‐bound beads with the cell lysis buffer, proteins were eluted by heating at 95°C for 5 min in the 2× sample buffer and they were separated by SDS–PAGE (8% SDS–PAGE gels).
5
Transfection and Protein Extraction Protocol
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Two million cells seeded 16–18 h prior to transfection in T75 flasks. 6 μg of appropriate expression construct or appropriate empty vector (control) were transfected with 24 μl Lipofectamine reagent (Life Technologies) and 12 μl Plus reagent (Life Technologies) (17 (link)). For coexpression experiments (Supplementary Figures S4 and S7), 4 μg of appropriate expression constructs were transfected separately and together, with empty vector used as filler to bring total DNA transfected to 8 μg. Approximately 24 h post-transfection, cells were washed 1× with phosphate buffered saline (PBS) and then harvested in 500 μl Total lysis buffer (TLB: 50 mM Tris, 150 mM NaCl, 10 mM EDTA, 0.5% SDS, 0.5% Triton-X, pH 7.2) supplemented with 10 μl/ml of Halt protease inhibitor cocktail, phosphate inhibitor cocktail 2, and phosphate inhibitor cocktail 3 (Sigma). After one round of freeze (−80°C) /thaw on ice, cells were sonicated with a Microson XL-2000 sonicator (Misonix) 3× (10 s sonication/10 s rest on ice), and cell lysates were then centrifuged at 4°C at 14 000 rpm for 15 min. Protein concentrations of cleared cell lysates were determined using BioRad protein assay (Bradford method).
6
Cytokine Profiling in Aortic Aneurysms
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Aneurysm tissues sampled 14 days after induction by the PPE model were lysed in Complete Mesoscale Lysis Buffer [10 mL 1x TRIS lysis buffer containing; 100 μL phosphate inhibitor cocktail #2 (Sigma-Aldrich, Søborg, Denmark), 100 μL phosphate inhibitor cocktail #3 (Sigma-Aldrich, Søborg, Denmark), and 1 tablet complete Mini, EDTA-free (Roche, Hvidovre, Denmark)] and homogenized by beads in a Tissue Lyzer II (Qiagen, Aarhus, Denmark) (Qiagen, Germany) for 3 min with maximum speed of 300 rpm and centrifuged for 20 min at +4°C at 12.000 × g. The supernatants were collected and stored at –80°C. Protein concentrations were determined using the Pierce BSA Protein Assay kit (Thermo Fischer, Roskilde, Denmark) (Bio-Rad, Copenhagen, Denmark). Cytokines and chemokines were then measured in aneurysmal protein samples and plasma samples using two different Mesoscale Discovery™ (MSD, Rockville, MA, United States) mouse pro-inflammatory V-Plex plus kits while TNFR1 and TNFR2 levels were determined using Ultra-Sensitive Kit (MSD, Rockville, MA, United States). Protocols were performed according to the manufacturer’s instructions. All samples were run in duplicate. Data were analyzed using MSD Workbench software. The cut-off threshold for inclusion of results was set at CV < 20%.
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