Proteins were extracted from RAW264.7 cells using radioimmunoprecipitation (RIPA) buffer (Yazyme, Shanghai). Protein concentration was determined by BCA protein detection kit (Biyuntian, Shanghai). SDS-PAGE was used to separate proteins from different samples. After transferring the separated proteins to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, CA, USA), the PVDF membranes were blocked with protein-free fast blocking solution (Yazyme, Shanghai) for 15 min. Blocked PVDF membranes were then incubated in diluted primary antibody for 12 h at 4°C. The dilution ratios of different antibodies (Boaosen, Beijing) were as follows: β-actin (1 : 1000), NF-κB-p65 (1 : 1000, Bioss), p-NF-κB-p65 (1 : 1000, Bioss), IL-1β (1 : 500, Bioss), IL-6 (1 : 500, Bioss), and TNF-α (1 : 500, Bioss). Next, the PVDF membrane and diluted secondary antibody were incubated for 1 h at room temperature. Finally, proteins on PVDF membranes were imaged using enhanced chemiluminescence (ECL) solution (GlpBio, USA), and protein bands were processed using ImageJ to detect protein expression levels.
Bca protein detection kit
Manufactured by Beyotime
Sourced in China, United States
The BCA protein detection kit is a colorimetric assay used to quantify the total protein concentration in a sample. It utilizes the bicinchoninic acid (BCA) method to measure the reduction of copper(II) ions to copper(I) ions by proteins in an alkaline environment. The resultant purple-colored complex can be measured spectrophotometrically, allowing for the determination of the protein concentration in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (23 mentions)
Ripa lysis buffer, by Beyotime (12 mentions)
Ripa buffer, by Beyotime (9 mentions)
Gapdh, by Abcam (6 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (4 mentions)
Ab9485, by Abcam (4 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Ab8245, by Abcam (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Ecl plus, by Thermo Fisher Scientific (3 mentions)
β actin, by Bioss Antibodies (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Ecl kit, by Merck Group (2 mentions)
Anti vimentin, by Abcam (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (2 mentions)
Immobilon nc gels, by Merck Group (2 mentions)
Protease, by Roche (2 mentions)
Protease and phosphatase inhibitor, by Beyotime (2 mentions)
Ab205718, by Abcam (2 mentions)
113 protocols using bca protein detection kit
1
Western Blot Analysis of Inflammatory Proteins
2
Occludin Expression in Colon Tissues
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Colon tissues were weighted and put into homogenizer to grind into tissue homogenate with mixture phenylmethylsulfonyl fluoride (PMSF) (Besbio, China) and radioimmunoprecipitation assay (RIPA) (Besbio, China). Protein concentration was measured by bicinchoninic acid assay (BCA) protein detection kit (Biyuntian, China) and micro-spectrophotometer. Proteins were electrophoresed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (12%) and transferred to nitrocellulose membranes. Membranes were blocked with skim milk (5%) in TBST for 1 h at room temperature and incubated with a rabbit anti-mouse polyclonal occludin antibody (diluted 1:1,000) (Proteintech, United States) at 4°C overnight (Zacharek et al., 2007 (link)). After being washed thrice in TBST, the membranes were reacted with a 1:1,000 dilution of horseradish peroxidase (HRP) goat anti-rabbit IgG (Dingguo Biotechnology Co., Ltd., China) for 1 h at room temperature. After being washed thrice in TBST, membranes were reacted with the mixture of peroxide solution (Bio-Rad, United States) and Luminol enhancer solution (Bio-Rad, United States) (1:1). Finally, the results of WB were detected by Tanon 5500 chemiluminescent imager system (Tianneng, China), referencing by β-actin protein as standardization. The process was repeatedly thrice for WB experiments.
3
Cell Metabolism Assays and Analyses
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MTT and DMSO were purchased from Tiangen Biochemical Technology Co., Ltd. (China). Glucose assay kit and glycogen staining kit and human insulin were purchased from Nanjing Jiancheng Co., Ltd. (China). High-glucose DMEM medium and phenol red-free 1640 medium and TRIzol and Lipofectamine 2000 were purchased from Thermo (USA). The PI3K inhibitor wortmannin S1952 was purchased from Biyuntian Biotechnology Company (China). Fetal bovine serum was purchased from Tianjin TBD Company (China). Trypsin was purchased from Promega (USA). cDNA first-strand synthesis kit and PCR mix and SYBR PCR mix were purchased from Takara (China). Protein extraction kit and BCA protein detection kit were purchased from Biyuntian Biotechnology Company (China).
4
Western Blot Analysis of Protein Samples
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Intestines or cells were lysed with RIPA lysate. The protein concentration was determined by a BCA protein detection kit (Biyuntian, China). Thirty grams of total protein was separated by 10% SDS–PAGE and transferred to a polyvinylidene fluoride membrane (Bio–Rad, USA). The membrane was blocked with 5% skim milk for 1 hour and then incubated with antibody at 4°C overnight. After three washes with TBST (10 minutes each), a secondary antibody (1:50,000, Abcam, UK) was incubated for 1 hour. After chemiluminescence, Image J software was used to quantify the gray values of the target band and internal reference (GAPDH).
5
Microglia Antioxidant Profile Analysis
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After microglia were treated with different factors, they were collected and homogenized in 500 μL PBS using ultrasonic cell crushers. Then, they were subjected to centrifugation at 4°C at 1200 RPM for 10 min to obtain the supernatant. After the experiment, 1.5 to 2.5 mL tail blood was extracted from each group and placed in a refrigerator at 4°C overnight. Then, the serum was gathered via centrifugation at 3000 RPM for 10 min. The rats’ brain tissues and normal saline were mixed at a ratio of 1:9. The mixture was homogenized on ice and centrifuged at 420 RPM for 10 min. The BCA protein detection kit (Beyotime) was employed to test the contents of MDA, SOD, CAT and GSH-Px in the cell supernatant and the concentration of MDA and SOD in the serum and brain tissue homogenates according to the regulations.
6
Macrophage Signaling Pathway Analysis
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Mice macrophages were prepared in 12-well plates as described above and incubated with and without peptides for 2 h at 37 °C. After 2 h incubation, cells were washed with Opti-MEM twice and infected with SC19 at a MOI of 5 for 0, 15, 30, 60, and 120 min to determine different protein expression. Then, the macrophages were lysed by the RIPA buffer with PMSF (Beyotime, Beijing, China) and concentrations were determined using a BCA protein detection kit (Beyotime). The cell lysates were separated by a 10–15% SDS-PAGE gel and subsequently transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were immunoblotted with anti-NF-κB p65 Ab (Bioss, Beijing, China), anti-phospho-NF-κB p65 Ab (Beyotime), anti-ERK1/2 Ab (Bioss), anti-phospho-ERK1/2 Ab (Cell signaling technology, Danvers, MA, USA), anti-TLR2 Ab (Wanlei Life Sciences, Shenyang, China), anti-TLR4 Ab (Santa Cruz Biotechnology, Inc.), and anti-β-actin antibody (Beyotime).
7
Protein Extraction and Western Blot Analysis
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Cell samples were lysed with cold RIPA lytic buffer (Beyotime, Shanghai, China) containing 1% PMSF (Beyotime, Shanghai, China). The extracted protein was quantified using BCA protein detection kit (Beyotime, Shanghai, China). After electrophoresis was performed on a 10% sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE), the blots were transferred to polyvinylidene difluoride (PVDF) membrane, which were further incubated with 5% skimmed milk diluted with Tris Buffered Saline with Tween (TBST) for 1 h. The membranes were incubated with diluted primary antibodies (details of antibodies are listed in Supplementary Table 2) at 4 °C overnight, and then with goat antirabbit or anti-mouse IgG polyclonal antibody labeled with horseradish peroxidase (HRP) for 1 h at room temperature. Immobilon Western Chemakum HRP Substrate (Milipore, MA, USA) was used for color development.
8
EGFR and MAPK Pathway Protein Analysis
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Total protein extraction was performed with RIPA lysis buffer (Beyotime). The BCA protein detection kit (Beyotime) was applied for protein quantification. Equal amounts of protein samples were detached on 10% SDS-PAGE, transferred onto PVDF membranes (Millipore, USA), and blocked in 5% defatted milk. PVDF membranes were subjected to overnight incubation with primary antibodies (all from Abacam, USA) for EGFR (ab52894, 1/1,000), phosphorylated EGFR (p-EGFR, ab40815, 1/500), MEK (ab32091, 1/1,000), phosphorylated MEK (p-MEK, ab96379, 1/1,000), ERK (ab32537, 1/1,000), phosphorylated ERK (p-ERK, ab131438, 1/500), and GAPDH (ab8245, 1/500) at 4°C and subsequently probed with HRP-labeled secondary antibodies (ab6721, 1/2,000) for 1.5 h at room temperature and detected on an enhanced chemiluminescence test system (Thermo Fisher Scientific, USA). GAPDH served as an internal reference.
9
Western Blotting Protein Analysis
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Western Blotting analysis were performed as described previously [32 (link)]. Briefly, the protein was isolated by RIPA lysis buffer (Beyotime) and the protein concentration was determined using the BCA protein detection kit (Beyotime), then transferred to PVDF membranes (Millipore, inc., USA) by SDS-PAGE. The membranes were blocked with 5% non-fat dry milk in TBS-T (20 mM Tris, 137 mM NaCl, 0.05% Tween-20, pH 7.4) at room temperature for 3 h and then incubated with primary antibodies at 4° C.
10
Comprehensive Protein Expression Analysis
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Initially, PC9 and A549 cells were treated with radioimmunoprecipitation assay (RIPA buffer, Beyotime, Shanghai, China) and quantitatively assessed by a bicinchoninic acid (BCA) protein detection kit (Beyotime, Shanghai, China). 10 µg of the extracted protein was loaded on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Sigma-Aldrich) and subjected to separation before being transferred onto polyvinylidene fluoride (PVDF) membranes. Further, 2.5% of skimmed milk was used to block the protein-blotted PVDF membranes. Afterward, the membranes were incubated with primary antibodies against DARS2 (1:1000), GAPDH (1:1000), AKT protein kinase B, (p-AKT 1:1000), p-AKT (1:1000), AKT (1:1000), phosphorylated-Phosphotylinosital 3 kinase (p-PI3K, 1:1000), and PI3K (1:1000) obtained from Abcam (Cambridge, USA). After incubation overnight, the horse-radish peroxidase (HRP)-conjugated secondary antibodies (1:1000, Abcam) were used to detect the protein blots. Finally, the protein signals were visualized through Clarity Western electrochemiluminescence (ECL) substrate (Bio-Rad, Hercules, CA, USA).
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