Total proteins were extracted from lung tissues or cells. Lung or cultured fibroblasts were lysed with radioimmunoprecipitation assay (RIPA) buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, and 50 mM Tris-HCl, pH 7.5) and centrifuged at 14,000 × g for 10 min at 4°C. Protein concentrations in supernatants were measured with a Bradford assay (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). Protein samples (20 µg/lane) were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. Membranes were blocked with 5% non-fat milk for 2 h at room temperature and incubated overnight at 4°C with the primary antibody against Col I, α-SMA, Fn, ACE and AT1 (all 1:500) followed by a peroxidase-labeled affinity-purified secondary antibody to rabbit/mouse IgG (1:5,000, cat. no. 074-1506, Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD, USA) for 2 h at 37°C. Target bands were visualized by the addition of an Enhanced Chemiluminescence Prime Western Blotting Detection reagent (GE Healthcare, Chicago, IL, USA). The density of specific bands was analyzed using Image-Pro Plus image processing software (version 6.0; Media Cybernetics, Inc., Rockville, MD, USA) and the results were normalized to β-actin or GAPDH (both 1:500).
Bradford assay
Manufactured by Solarbio
Sourced in China
The Bradford assay is a spectrophotometric analytical procedure used to measure the concentration of protein in a solution. It is a widely used method for quantifying protein levels in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescence prime western blotting detection reagent, by GE Healthcare (1 mentions)
Ab271136, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Ab37168, by Abcam (1 mentions)
Imagej software, by Bio-Rad (1 mentions)
8 plex itraq reagent, by Thermo Fisher Scientific (1 mentions)
Strata x c18 column, by Phenomenex (1 mentions)
Ultremex scx column, by Phenomenex (1 mentions)
3 protocols using bradford assay
1
Quantifying Lung Fibrosis Proteins
2
Protein Extraction and Quantification
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Cell pellets were lysed with radioimmunoprecipitation lysis (Solarbio) on ice for 30 min, followed by centrifugation at 12,000 g at 4°C for 10 min. Protein concentration was assayed with Bradford assay (Solarbio), and extracted protein was resolved through SDS‐PAGE and transferred to polyvinylidene difluoride membranes (Millipore, USA). The membranes were sealed by 5% BSA in TBST, followed by incubation with primary antibodies of KIAA1429 (1/1000; ab271136; Abcam), GAPDH (1/2000; ab37168), SLC7A11 (1/2000; ab175186), FSP1 (1/1000; ab197896), DHODH (1/1000; ab174288), GPX4 (1/1000; ab252833) or ACSL4 (1/10000; ab155282) along with secondary antibodies (1/10000; 31460; Thermo Fisher). The immunoreactivity was captured through chemiluminescence imaging system (Solarbio). Afterwards, optical density was quantified with ImageJ software (Bio‐Rad).
3
Protein Preparation and Fractionation
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Samples were diluted in lysis buffer (7 mol/L urea, 2 mol/L thiourea, 4% CHAPS, and 40 mmol/L Tris-HCl, pH 8.5) and reduced with 10 mmol/L DTT (final concentration after mixing) at 56°C for 1 h. The samples were then alkylated with 55 mmol/L IAM (final concentration) in a dark room for 1 h. After reduction and alkylation, protein mixture was precipitated by adding 4x volume of chilled acetone and staying still at −20°C overnight. After centrifuging at 30000g at 4°C, the protein pellet was collected. Then, the pellet was dissolved in 0.5 M TEAB (Applied Biosystems, Milan, Italy) and sonicated in ice. After centrifuging at 30,000g at 4°C, an aliquot of the supernatant was taken for determining protein concentration with Bradford assay (Solarbio, Beijing, China) [9 (link)]. Proteins (100 μg) from each sample were typically digested and labeled with 8-plex iTRAQ reagents (Applied Biosystems, USA).
The labeled samples were pooled and eluted into 20 fractions with an Ultremex SCX column containing 5 μm particles (Phenomenex, USA). The eluted fractions were then desalted with a Strata X C18 column (Phenomenex, USA) and dried under vacuum [10 (link)].
The labeled samples were pooled and eluted into 20 fractions with an Ultremex SCX column containing 5 μm particles (Phenomenex, USA). The eluted fractions were then desalted with a Strata X C18 column (Phenomenex, USA) and dried under vacuum [10 (link)].
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