Total protein in atherosclerotic plaque samples was extracted using the Solarbio protein extraction kit (BC3710); concentrations were quantified by a BCA protein assay kit (LABLEAD, B5000-500T). For immunoblotting, equal amounts of proteins were separated on an 4% to 12% SDS-PAGE gel and electrophoretically transferred to a polyvinylidene difluoride microporous membrane (0.22 μm pore size; Beyotime) and blocked with 5% non-fat milk for 1 h at room temperature, then blotted with primary antibody (Supplementary Table S2 ) of targeted proteins overnight at 4 ℃. After incubation with HRP-conjugated secondary antibody for 1 h at room temperature, the membranes were developed using the PierceTM ECL Western Blotting Substrate (Thermo Scientific; Cat: 32,209). Quantitative analysis was carried out by Bio‐Rad ChemiDoc XRS image analysis system according band density. Restore Western Blot Stripping Buffer (APPLYGEN; Cat: P1651) was used to strip membranes when it’s needed.
Chemidoc xrs image analysis system
Manufactured by Bio-Rad
Sourced in United States
The ChemiDoc XRS image analysis system is a versatile lab equipment designed for capturing and analyzing images of electrophoresis gels, Western blots, and other samples. It features a high-resolution camera and advanced imaging software for researchers to document and quantify their experimental results.
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4 protocols using chemidoc xrs image analysis system
1
Quantifying Protein Levels in Atherosclerotic Plaque
2
Rat ADSC Protein Profiling by Western Blot
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Rat ADSCs in the fourth passage were harvested by trypsinization, and 1×106 cells/ml were fixed in 100 µl protein isolation, followed by centrifugation at 12,000 g for 30 min at 4°C. The cell lysates were analyzed using a commercial bicinchoninic acid assay kit (cat. no. BCA01; Beijing Dingguo Biotech, Co., Ltd. Beijing, China). A total of 30 µg protein was prepared per sample, and denatured at 95°C for 5 min. The proteins were resolved by 15 or 12% SDS-PAGE (Beijing Dingguo Biotech, Co., Ltd.) and transferred onto PVDF membranes. The membranes were blocked in 5% bovine serum albumin for 1 h, and then incubated overnight at 4°C with GFAP, S100, P75 and GAPDH primary antibodies (1:10,000, Proteintech Group, Inc.). The membranes were then incubated with goat anti-mouse (cat. no. L3032-1/2) and anti-rabbit (cat. no. L3012-1/2) IgG-horseradish peroxidase-conjugated secondary antibodies (1:10,000; Signalway Antibody, College Park, MD, USA) for 1 h at 37°C. The blots were scanned using a ChemiDoc XRS+ image analysis system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and analyzed using Image J software (National Institutes of Health, Bethesda, MD, USA).
3
Protein Expression Analysis in Cell Lysates
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To assess the total protein content, 1 mM phosphatase inhibitor cocktail and 1 mM PMSF were added to each sample in a RIPA lysate buffer. Using the BCA test technique, the total protein concentration of each sample was measured, and each sample's concentration was corrected to match. Samples of protein were combined with a loading buffer of 5 × , denatured at 95 °C, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then electrophoretically transferred onto membranes made of polyvinylidene fluoride. These membranes were probed with rabbit anti-p-PI3 Kinase (1:1000; CST®; #17366S), rabbit anti-PI3 Kinase (1:1000; CST®; Cat. No. #4249T), rabbit anti-p-Akt (1:1000; CST®; #4060S), rabbit anti-Akt (1:1000; CST®; #4691S), rabbit anti-p-mTOR (1:1000; CST®; #2983S), rabbit anti-mTOR (1:1000; CST®; #5536S), rabbit anti-GR (1:1000; CST®; #12041S) and rabbit anti-β actin (1:1000; Servicebio®; #GB113225) antibodies overnight at 4 °C, anti-rabbit IgG HRP-conjugated antibody (1:3000; Servicebio®; #GB23303) was added the next day at indoor temperature for 80 min. A Chemi-Doc XRS + image analysis system (BioRad, USA) was used to measure total protein.
4
Quantitative Analysis of FOS Protein
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We extracted PVAT sample protein using the Solarbio protein extraction kit (BC3710). A BCA protein assay kit (LABLEAD, B5000-500T) was used to determine protein concentration. Equal amounts of PVAT protein were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes. After blocking with 5% nonfat milk for 1 h, we incubated membranes with primary antibodies to FOS (1:5000, Proteintech; 66590-1-lg) overnight at 4°C. Membranes then were incubated with HRP-conjugated secondary antibody for 1 h at room temperature. We used PierceTM ECL Western Blotting Substrate (Thermo Scientific; Cat: 32209) to detect immunoreactive bands. Bio‐Rad ChemiDoc XRS image analysis system (Bio‐Rad) was used to perform quantitative analysis according to band density.
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