The liver microsomes of rats given sesamin were prepared according to the modified methods of previous report (Naritomi et al. 2001 (link)). Briefly, the liver was excised, rinsed with ice-cold saline solution, and homogenized with four volumes of ice-cold 1.15% KCl. The homogenate was centrifuged at 10,000g for 20 min, and the supernatant was then centrifuged at 100,000g for 60 min. The pellet was reconstituted with 20 mmol/L phosphate buffer (pH 7.4) containing 1.15% KCl, and the protein concentration was determined using Protein Assay Bicinchoninate Kit (Nacalai tesque, Inc., Kyoto, Japan).
Protein assay bicinchoninate kit
Manufactured by Nacalai Tesque
Sourced in Japan
The Protein Assay Bicinchoninate Kit is a laboratory reagent used for the quantitative determination of protein concentration in samples. The kit utilizes the bicinchoninic acid (BCA) method to measure the total protein content. It provides a simple, colorimetric detection and quantitation of protein in solution.
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Lab products found in correlation
Blocking one, by Nacalai Tesque (6 mentions)
Immuno blot pvdf membrane, by Bio-Rad (3 mentions)
C digit, by LI COR (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Complete protease inhibitor cocktail, by Nacalai Tesque (2 mentions)
Proteinase inhibitor cocktail, by Nacalai Tesque (2 mentions)
Pierce protease and phosphatase inhibitor, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Nacalai Tesque (2 mentions)
Ripa buffer, by Nacalai Tesque (2 mentions)
Arc 7010, by Hitachi (2 mentions)
Suc leu leu val tyr 4 methylcoumaryl7 amide mca, by Peptide Institute (2 mentions)
Chemi lumi one ultra reagent, by Nacalai Tesque (1 mentions)
A1978, by Merck Group (1 mentions)
Ab109199, by Abcam (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
Ud 100, by TOMY (1 mentions)
Chemidoc xrs imager, by Bio-Rad (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Westar supernova, by Cyanagen (1 mentions)
Complete mini, by Roche (1 mentions)
34 protocols using protein assay bicinchoninate kit
1
Rat Liver Microsome Preparation
2
Protein Extraction and Western Blot Analysis
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Total protein from the C2C12 cells was extracted with a lysis buffer containing 50 mM HEPES (pH: 7.6), 150 mM NaCl, 10 mM EDTA, 10 mM Na4P2O7, 10 mM NaF, 2 mM Na3VO4, 1% (vol/vol) NP-40, 1% (vol/vol) Na-deoxycholate, 0.2% (wt/vol) SDS, and 1% (vol/vol) complete protease inhibitor cocktail (Nacalai Tesque Inc. Kyoto, Japan). Protein concentrations were measured using a Protein Assay Bicinchoninate Kit (Nacalai Tesque Inc. Kyoto, Japan). Before SDS-PAGE, an aliquot of the extracted protein solution was mixed with an equal volume of the sample loading buffer containing 1% (vol/vol) 2-mercaptoethanol, 4% (wt/vol) SDS, 125 mM of Tris-HCl (pH: 6.8), 10% (wt/vol) sucrose, and 0.01% (wt/vol) bromophenol blue. The mixture was then heated to 37℃ for 30 min. Then, 10 mg of protein was separated on SDS-polyacrylamide gels and electrically transferred to an ImmunoBlot PVDF membrane (Bio-Rad Laboratories, Hercules, CA, USA). The blot was blocked with the Blocking One (Nakalai Tesque Inc. Kyoto, Japan) for 1 h at room temperature and incubated with primary antibodies overnight at 4℃ in TBS containing 0.1% Tween-20. The signals were detected using the Immunostar Zeta or LD (Wako Chemicals. Osaka, Japan), quantified using the C-Digit (LI-COR Biosciences, Lincoln, NE, USA), and expressed as arbitrary units.
3
Western Blot Analysis of Cellular Proteins
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Protein lysate was prepared from cultured cells by using an ice-cold cell lysis buffer supplemented with a protease inhibitor cocktail (Roche; Basel, Switzerland). Protein concentration was measured with a protein assay bicinchoninate kit (Nacalai Tesque, Kyoto, Japan). Western blot analysis was performed as previously described [45 (link)]. Detection was performed by using Chemi-Lumi One Ultra Reagent (Nacalai Tesque). The dilution of primary antibodies used was as follows: anti-CA9 (1:2000), anti–HIF–1α (1:1000), anti-transferrin receptor-1 (1:2000), anti-IRP1/IRP2 (1:2000), anti-ferritin light/heavy chain (1:1000), anti-SLC40A1 (1:500), anti-LC3B (1:1000), anti-LAMP-1 (1:1000), anti-cleaved caspase-3 (1:500) and anti-β-actin (1:2000).
4
Protein Expression Analysis in Tissues
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Frozen tissues were homogenized and lysed in radioimmunoprecipitation assay buffer containing 0.1% sodium dodecyl sulfate and 1% proteinase inhibitor cocktail (Nacalai Tesque) on ice. Protein concentrations were determined using a Protein Assay Bicinchoninate kit (Nacalai Tesque). Lysates were electrophoresed on Mini-PROTEAN TGX gels (Bio-Rad Laboratories, Inc., Tokyo, Japan) and then electrophoretically transferred to Immobilon-P polyvinylidene difluoride membranes (Merck Millipore, Burlington, MA). Immunoblotting was performed using specific antibodies against β-actin (A1978, 1:1000, Sigma-Aldrich, St. Louis, MO) and p21 (ab109199, 1:500, Abcam), as described previously.19 (link)
5
SDS-PAGE Protein Extraction and Analysis
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Protein extraction and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) were performed according to the previously established protocol [33 (link)]. Cell line samples were lysed using the RIPA buffer (150 mM NaCl, 50 mM Tris–HCl (pH 7.4), 1% Nonidet P-40, 1% SDS, 0.5% deoxycholate), containing PierceTM Protease and Phosphatase inhibitor (Thermo Scientific). Subsequently, they were homogenized by super-sonication (UD-100, TOMY, Tokyo, Japan) and clarified by centrifugation at 18,000 × g for 30 min at 4 °C. The total protein concentration of the lysates was quantified using the Protein Assay Bicinchoninate kit (Nacalai tesque, Kyoto, Japan).
6
Protein Extraction and Western Blot Analysis
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Cells were harvested in RIPA buffer (150 mM sodium chloride, 1% Triton-X, 50 mM Tris, pH 8.0, 10 mM EDTA, pH 13, 10 mM PMSF, PIC). Protein quantification was performed using the Protein Assay Bicinchoninate Kit (Nacalai Tesque, Japan) and 2 mg/ml BSA (Thermo Fisher Scientific) was used to construct the standard curve. The purified protein lysates were subjected to SDS-PAGE followed by Western Blotting. The blots were developed using the WesternBright enhanced chemiluminescent HRP substrate (Advansta, CA, United States) or Westar Supernova (Cyanagen, Italy) and the bands were captured with the ChemiDoc XRS imager (BioRad).
7
Quantitative Analysis of ACE2 and TMPRSS2
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Cells were lysed with a lysis buffer (250 mM NaCl, 50 mM Tris pH 7.4, 50 mM NaF, 0.1 mM NaVO4, 5 mM EDTA, 0.1% Triton-X) including Complete Mini, EDTA-free (Roche, Basel, Switzerland) and PhosStop (Roche). Tissues were homogenized, sonicated, and lysed with the same lysis buffer. Protein concentration was quantified, using Protein Assay Bicinchoninate kit (06385-00; Nacalai Tesque, Kyoto, Japan).(12 (link)) SDS-PAGE electrophoresis was carried out by loading 20 μg protein. Blots were incubated with primary antibodies diluted in Can Get Signal® Solution 1 (TOYOBO, Osaka, Japan) overnight at 4°C, followed by horseradish peroxidase-conjugated secondary antibody (P0448; DAKO, Santa Clara, CA) diluted 1:10,000 in Can Get Signal® Solution 1 with incubation for 1 h at room temperature. Reactions on the membranes were finally visualized by Chemi-Lumi One Super (02230-30; Nacalai) and analyzed with ImageJ software. Anti-ACE2 (1:1,000 dilution), anti-TMPRSS2 (1:2,000 dilution) and anti-GAPDH (1:10,000 dilution) were used, respectively.
8
Immunoblotting Analysis of Cellular Proteins
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Immunoblotting analysis was performed as described previously with some modifications61 (link). Ly-6G+ cells or HUVECs were lysed in RIPA buffer (Nacalai Tesque, Inc., Kyoto, Japan) or Nonidet P-40 (NP-40) lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40), respectively. These buffers were supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail (Nacalai Tesque, Inc., Kyoto, Japan). The protein concentrations of the samples were determined using a Protein Assay Bicinchoninate Kit (Nacalai Tesque, Inc., Kyoto, Japan). Samples were applied to 10% polyacrylamide gels containing sodium dodecyl sulfate, subjected to electrophoresis, and transferred to a polyvinylidene difluoride membrane (Immobilon P; Millipore, MA, USA). The membrane was blocked with Blocking One or Blocking One-P (Nacalai Tesque, Inc., Kyoto, Japan). The proteins were immunoblotted with each antibody.
9
Iodinated Amino Acid Uptake Assay
10
Bronchoalveolar Lavage Fluid Analysis
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BAL was performed 10 min after PLY administration. One milliliter of PBS containing 2 mM EDTA was infused into the lungs through the intubated catheter, and 700 μL of the solution was collected as BAL fluid. The total protein concentration, albumin concentration, and LDH activity in the BAL fluid were measured using Protein Assay Bicinchoninate kit (nacalai), Mouse Albumin ELISA Quantitation Set (Bethyl Laboratories), and LDH Cytotoxicity Detection Kit (Takara), respectively, according to the manufacturers’ protocols. The concentrations of eicosanoids were measured by LC-MS/MS as described previously21 (link). The PC concentration was measured in an enzyme-based fluorescent assay as previously reported18 (link).
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