For the isolation of total cholesterol (TC), 1 × 106/mL LX-2 cells were lysed by 0.1% NP40 lysis buffer and then lipids were extracted with chloroform:isopropanol (7:11) in a micro-homogenizer. The sample was centrifuged at 13,000× g for 10 min and then air-dried at 50 °C to remove residual organic solvents. The pellets were dissolved in the Cholesterol Assay Buffer (Sigma, St. Louis, MO, USA) and vortexed until the mixture was homogenous. For the isolation of triglycerides (TG), 1 × 106/mL LX-2 cells were lysed by IGEPAL CA-630 (Beyotime, Shanghai, China). The sample was slowly heated to 100 °C in a metal bath for 3 min and then cooled to room temperature (RT). Centrifugation took place at 16,000× g for 2 min to collect the supernatant. The amounts of TG, TC, and FC were measured using a cholesterol test kit (Sigma, St. Louis, MO, USA) and triglyceride detection kit (Sigma, St. Louis, MO, USA), respectively, according to the manufacturer’s instructions. Protein concentrations were quantified by the BCA protein quantitative analysis kit (ThermoFisher, Waltham, MA, USA).
Bca protein quantitative analysis kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The BCA protein quantitative analysis kit is a colorimetric detection and quantitation assay used to measure the total protein concentration in a sample. The kit utilizes the bicinchoninic acid (BCA) reagent, which reacts with protein to produce a purple-colored complex that absorbs light at 562 nm. This allows for the quantification of protein levels in a sample by comparing the sample's absorbance to that of a standard curve.
Automatically generated - may contain errors
Lab products found in correlation
Cholesterol test kit, by Merck Group (1 mentions)
Igepal ca 630, by Beyotime (1 mentions)
Atp detection buffer, by Beyotime (1 mentions)
Cytation 3, by Agilent Technologies (1 mentions)
Nitrocellulose nc membrane, by Merck Group (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Beyotime (1 mentions)
Infinite 200, by Tecan (1 mentions)
Hydrogen peroxide assay kit, by Beyotime (1 mentions)
P c raf, by Cell Signaling Technology (1 mentions)
P src, by Cell Signaling Technology (1 mentions)
C raf, by Cell Signaling Technology (1 mentions)
P mek, by Cell Signaling Technology (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Sds page gel preparation kit, by Beyotime (1 mentions)
Tsg101, by Cell Signaling Technology (1 mentions)
Atg13, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
7 protocols using bca protein quantitative analysis kit
1
Cholesterol and Triglyceride Quantification
2
ATP Quantification in LX-2 Cells
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The LX-2 cells (5 × 105) were seeded into each well of a 6-well plate. After PA-retinol incubation or depletion, the cells were lysed by adding 200 μL ATP detection lysis buffer (Beyotime, Shanghai, China) and they were centrifuged at 4 °C and 12,000× g for 5 min. After transferring 100 μL of ATP detection buffer (Beyotime, Shanghai, China) to a 96-well plate, incubation took place for 5 min at RT and 20 μL cell lysate supernatant was added. Relative light units (RLU) were detected by a chemiluminescence instrument (Cytation3, Biotek, Winooski, Vt, USA). Protein concentration was determined with the BCA protein quantitative analysis kit (ThermoFisher, Waltham, MA, USA).
3
Western Blot Analysis of Proteins
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Proteins were extracted from the cell pellet by RIPA buffer comprised of protease and phosphatase inhibitors. The concentration of the extracted proteins was measured using the BCA Protein Quantitative Analysis Kit (Thermo, MA, USA, China). A total of 20 µg lysates were loaded onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to nitrocellulose (NC) membranes (Sigma, USA). The membranes were blocked with 5% skim milk for 2 hours, and subsequently incubated with primary antibodies overnight. After incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies for 3 hours, protein bands were detected with Immobilon Western Chemiluminescent HRP Substrate (Beyotime Biotechnology, China). Densitometric quantification was carried out using ImageJ software version 1.46r (ImageJ, USA).
4
Oxidative Stress Biomarkers in Lung Tissue
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Superoxide dismutase (SOD, A001-3), catalase (CAT, A007-1-1) and glutathione peroxidase (GPx, A005) activities, as well as malondialdehyde (MDA, A003-1) levels in lung tissue sample were determined by commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer's instructions. H2O2 levels in lung tissues were measured using a hydrogen peroxide assay kit, (Beyotime Institute of Biotechnology, Haimen, China) following the manufacturer's instructions. O2− in lung tissues was measured through lucigenin chemiluminescence method. Briefly, lung tissues of mice under different conditions were weighed and homogenized in a homogenization buffer using HEPES and EDTA. Following centrifugation (1,000 × g at 4°C for 10 min), an aliquot of the supernatant was incubated with 5 µM lucigenin in Krebs-HEPES buffer. Light emission was measured with a Tecan Infinite 200 (Tecan Group AG, Männedorf, Switzerland). Specificity for O2− was evaluated by adding SOD (350 U/ml) to the incubation medium. BCA Protein Quantitative Analysis kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used to measure the protein concentration.
5
Protein Expression Analysis of Signaling Cascades
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Lung tissues or neutrophils were lysed with RIPA buffer containing protease inhibitor and phosphatase inhibitor for protein collection. Then, the protein concentration was quantified using BCA Protein Quantitative Analysis Kit (ThermoScientific, Rockford, IL, USA). Appropriate amount of protein samples was separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to PVDF (polyvinylidene fluoride) membrane. Membranes were blocked with 5% skimmed milk for 1 h and incubated overnight at 4°C with primary antibodies: Src, P-Src, c-Raf, P-c-Raf, MEK, P-MEK, ERK, and P-ERK (Cell Signal Technology, Beverly, MA, USA, diluted at 1 : 1000). After washing with TBST buffer, membranes were incubated with goat anti-mouse IgG-HRP and goat anti-rabbit IgG-HRP (Engibody Biotechnology, Milwaukee, WI, USA, diluted at 1 : 2000), for 2 h at room temperature. The blots were imaged by electrochemiluminescence (ECL) detection kit (Pierce, Rockford, IL, USA) and Imaging System (Bio-Rad, USA).
6
Protein Expression Analysis in NRK-52E Cells
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NRK-52E cells were seeded in 6-well plates. The total protein of cells was extracted using a protein extraction kit (KeyGEN BioTECH, Nanjing, China). The concentration of protein was measured using a BCA protein quantitative analysis kit (ThermoFisher, USA). Then, the protein samples were mixed with loading buffer (Beyotime, Shanghai, China), and the protein was denatured by boiling.
The SDS-PAGE gel preparation kit (Beyotime, Shanghai, China) was used to prepare the gels of appropriate concentration based on the relative molecular mass of the protein. After the electrophoresis was completed, the protein was transferred to polyvinylidene fluoride (PVDF) membranes (ThermoFisher, USA). The membranes were incubated with 5% skim milk to block non-specific antigens, followed by incubation with the primary antibodies (Bax, Bcl-2, LC3, ATG13, CD63, Tsg101, AKT, p-AKT and GAPDH, all from Cell Signaling Technology, USA). Afterwards, the membranes were incubated with the secondary antibody. Finally, the protein blots were developed using the Molecular Imager ChemiDoc XRS + System.
The SDS-PAGE gel preparation kit (Beyotime, Shanghai, China) was used to prepare the gels of appropriate concentration based on the relative molecular mass of the protein. After the electrophoresis was completed, the protein was transferred to polyvinylidene fluoride (PVDF) membranes (ThermoFisher, USA). The membranes were incubated with 5% skim milk to block non-specific antigens, followed by incubation with the primary antibodies (Bax, Bcl-2, LC3, ATG13, CD63, Tsg101, AKT, p-AKT and GAPDH, all from Cell Signaling Technology, USA). Afterwards, the membranes were incubated with the secondary antibody. Finally, the protein blots were developed using the Molecular Imager ChemiDoc XRS + System.
7
Western Blot Analysis Procedure
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The cells were lysed in RIPA lysis buffer (Beyotime, Shanghai, China) with 1 mM PMSF and 10 ug/mL complete protease inhibitor mixture (Roche, Basel, Switzerland). The protein concentration was measured by a BCA protein quantitative analysis kit (ThermoFisher, Waltham, MA, USA). The protein samples were separated by 10% SDS-PAGE gel electrophoresis and then transferred to a PVDF membrane (Merck, Kenilworth, NJ, USA). The membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) and incubated with primary antibodies (Table S2 ) at 4 °C overnight. After washing in TBST, the membranes were further incubated with a horseradish peroxidase-labeled goat anti-rabbit antibody (Cell Signaling Technology, Danvers, MA, USA). The protein bands were captured using the ChemiDocTM MP Imaging System (Bio-Rad, Hercules, CA, USA) and quantified with Image J Software (Version 1.51K).
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