Western blot analysis was conducted as previously described (Meng et al., 2014 (link)). Right cortex tissues were collected from each rat (n = 3), and the total protein was extracted using a protein extraction reagent supplemented with protease and phosphatase inhibitor cocktails (ComWin Biotech, China). The total protein concentration of each sample was determined by a BCA kit (ComWin Biotech, China). Equal amounts of protein were separated using SDS-polyacrylamide gel electrophoresis and were transferred to nitrocellulose membranes. These membranes were blocked before being incubated overnight at 4°C with the appropriate primary antibodies: cnpase (ab183500, 1:1000), MBP (ab209328, 1:1000), Vimentin (ab92547, 1:10,000), SYP (ab32127, 1:1000), PSD95 (ab76115, 1:1000), MAP-2 (ab32454, 1:2000), Tau-1 (ab75714, 1:1000), BDNF (ab108319, 1:1000), p-TrkB (Tyr705) (ab229908, 1:1000), TrkB (CST4603, 1:1000), p-CREB (Ser133) (ab32096, 1:1000), CREB (ab32515, 1:1000), p-Akt (Ser473) (CST4060, 1:1000), Akt (CST4685, 1:1000), and β-actin (EXP0036 F, 1:2000). Then, the membranes were washed three times and incubated with the appropriate secondary antibodies. The blots were visualized using enhanced chemiluminescence western blot detection kits (ComWin Biotech, China). The blot densities were calculated by ImageJ software.
Bca kit
Manufactured by CoWin Biotech
Sourced in China
The BCA kit is a laboratory reagent used for the quantitative determination of total protein concentration. It utilizes the bicinchoninic acid (BCA) assay, a colorimetric detection method, to measure the protein content in samples. The kit provides a simple, reliable, and accurate means of protein quantification.
Automatically generated - may contain errors
Lab products found in correlation
Protease and phosphatase inhibitor cocktail, by CoWin Biotech (3 mentions)
Enhanced chemiluminescence western blot detection kits, by CoWin Biotech (2 mentions)
Gel image analysis system, by Bio-Rad (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Proteintech (1 mentions)
Polyvinylidene difluoride pvdf membrane, by Merck Group (1 mentions)
Radio immunoprecipitation assay ripa lysis buffer, by Beyotime (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Gadph, by Proteintech (1 mentions)
Fetal bovine serum (fbs), by Sartorius (1 mentions)
High glucose dmem, by Thermo Fisher Scientific (1 mentions)
Mmp detection kit, by Beyotime (1 mentions)
Lc3 antibody, by Abcam (1 mentions)
Beclin 1, by Proteintech (1 mentions)
Anti myd88, by Abcam (1 mentions)
Anti il 1β, by Abcam (1 mentions)
Anti il 6, by Abcam (1 mentions)
Anti nf κb, by Cell Signaling Technology (1 mentions)
Anti β actin, by Proteintech (1 mentions)
Rabbit anti gapdh, by Affinity Biosciences (1 mentions)
Rabbit anti nur77, by Proteintech (1 mentions)
11 protocols using bca kit
1
Western Blot Analysis of Neurobiological Markers
2
TPCN2 Protein Expression Analysis
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The cells were lysed with a Radio-Immunoprecipitation Assay (RIPA) lysis buffer (Beyotime Biotechnology, Shanghai, China) containing protease and phosphatase inhibitors (NCM Biotech, Suzhou, China). The protein concentration of the cells was determined with a bicinchoninic acid [BCA] kit (ComWin Biotech, Beijing, China), and 40 μg of protein was prepared for a sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride [PVDF] membrane (Millipore, Burlington, MA, USA). The membranes were blocked with 5% nonfat dry milk for 1 h, and then incubated with rabbit anti-TPCN2 monoclonal antibodies (1:200; Alomone Labs, Jerusalem, Israel) overnight at 4°C. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:8000; Proteintech, Rosemont, IL, USA) was used as the loading control. The results were visualized using a gel image analysis system (Bio-Rad, Hercules, CA, USA) as per the manufacturer's instructions.
3
Protein Expression Profiling in Neuroinflammation
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Right cortex tissues, serum and cell lysis solution were collected from each sample, and the total protein was extracted using a protein extraction reagent supplemented with protease and phosphatase inhibitor cocktails (ComWin Biotech, Beijing, China). The total protein concentration of tissue and cell samples was determined by a BCA kit (ComWin Biotech, Beijing, China). The expression level of TNF-α, IL-1β, IL-6, MCP-1, CD11a, ICAM-1, caspase-1, NOX, PC, 4-HNE and 8-OHdG was assessed using enzyme-linked immunosorbent assay (ELISA) kits according to the previous method (Xie et al. 2019 ). All ELISA kits were purchased from Expandbio (Beijing, China).
4
Protein Expression Analysis in Rat Cortex
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Western blot analysis was conducted as previously described (Xie et al. 2019 ; Zhu et al. 2021d (link)). Right cortex tissues were collected from each rat (n = 3), and the total protein was extracted using a protein extraction reagent supplemented with protease and phosphatase inhibitor cocktails (ComWin Biotech, Beijing, China). The total protein concentration of each sample was determined by a BCA kit (ComWin Biotech, Beijing, China). Equal amounts of protein were separated using SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked before being incubated overnight at 4 °C with the appropriate primary antibodies against the following proteins: NLRP3 (A5652, 1:1000), IL-1β (Ab16288, 1:1000), Nrf2 (A0674, 1:1000), HO-1 (A1346, 1:1000), Akt (CST4685, 1:1000), p-Akt (CST4060T, 1:1000) and β-actin (EXP0036 F, 1:2000). Then, the membranes were washed three times and incubated with the appropriate secondary antibodies. The blots were visualized using enhanced chemiluminescence western blot detection kits (ComWin Biotech, Beijing, China). The blot densities were measured by Image J software (Bethesda, MD).
5
Evaluation of Oxidative Stress and Autophagy
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K2Cr2O7 was acquired from Kaitong Chemical (Shanghai, China). Total LRMA was a gift from Professor Ding Xinhua, School of Plant Protection, Shandong Agricultural University. DMEM/high glucose and Trypsin were purchased from Gibco Company (Grand Island, NE, USA). Fetal bovine serum was purchased from Biological Industries (State of Israel). MMP detection kit, ROS (DCFH-DA), and TOMM20 antibody were supplied from the Beyotime Institute of Biotechnology (Haimen, Jiangsu, China). LC3 antibody was purchased from Abcam. p62, Beclin-1, ATG5, and GADPH antibodies were obtained from Proteintech (Chicago, IL, USA). Parkin antibody (A0968) was purchased from ABclonal (Wuhan, China). Chicken PINK1 ELISA kits were purchased from Enzyme-linked Biotechnology (Shanghai, China). BCA kit was bought from ComWin Biotech (Beijing, China).
6
Western Blot Analysis of Inflammatory Signaling in Lung Tissues
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Lung tissues were homogenized in the 4°C RIPA buffer (Beyotime Institute of Biotechnology, Shanghai, China) to obtain total protein. The protein concentration was determined using a BCA kit (ComWin Biotech, China). Subsequently, the protein samples were loaded and electrophoresed on SDS-polyacrylamide gels and were transferred onto membranes (Millipore, MA, United States). The membranes were blocked with a blocking solution (5% skim milk) for 1 h at room temperature and incubated with the following primary antibodies overnight at °C: anti-NLRP3 (dilution 1:1,000, Abcam 263899), anti-Caspase-1 (dilution 1:1,000, Abcam 108362), anti-TLR4 (dilution 1:1,000, Abcam 22048), anti-My-D88 (dilution 1:1,000, Abcam 2064), anti-NF-κB (dilution 1:1,000, Cell Signaling Technology 8242), anti-IL-1β (dilution 1:1,000, Abcam 9722), anti-IL-6 (dilution 1:1,000, Abcam 233706), anti-GSDMD (dilution 1:1,000, ab219800) and anti-β actin (dilution 1:4,000, Proteintech Group). The next day, all membranes were incubated with a secondary antibody conjugated to horseradish peroxidase at room temperature for 1 h. Finally, the membranes were imaged using enhanced chemiluminescence (ComWin Biotech, China) using a Bio-Rad ChemiDoc gel system.
7
Protein Extraction and Western Blot Analysis
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After 48 h of cell treatment, a protein lysis buffer was used to extract the total protein and the protein concentration was determined using a BCA kit (CoWin Biosciences, Beijing). Following the addition of the same amount of protein, polyacrylamide gel electrophoresis was implemented using 100 g/L sodium lauryl sulfate. Once the electrophoresis was completed, the protein was transferred onto a 0.45 μm PVDF membrane, which was then sealed in a confining liquid. The phosphorylated and nonphosphorylated proteins were sealed using 5% fetal bovine serum and 50 g/L skimmed milk powder, respectively. After sealing, the primary and secondary antibodies were added accordingly. This point represents the completion of the method development. The primary antibodies used here were as follows: rabbit anti‐GAPDH (1:5000, Affinity, USA); rabbit anti‐Nur77 (1:1000, Proteintech, USA); and rabbit anti‐CXCR4, rabbit anti‐PI3K and p‐PI3K, AKT, and p‐AKT (all 1:1000, Abcam, UK). The secondary antibody used here was goat anti‐rabbit IgG (1:1000, CST, USA). Each experiment was repeated in triplicate.
8
Western Blot Analysis of HMGB1, TLR4, and NF-κB
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Proteins were extracted from A549 cells using radioimmunoprecipitation assay buffer with protease inhibitor cocktail (both Cell Signaling Technology, Inc.). Protein concentration was determined using a BCA kit (CoWin Biosciences). The proteins (15 µg/lane) were separated using 10% SDS-PAGE and transferred to nitrocellulose membranes (Amersham; Cytiva). The membranes were subsequently blocked with 5% skim milk at room temperature for 2 h. Proteins were detected by western blotting using rabbit anti-HMGB1 (1:400; cat. no. ab18256; Abcam), rabbit anti-Toll-like receptor 4 (TLR4; 1:400; cat. no. ab13556; Abcam), rabbit anti-NF-κB p65(1:400; cat. no. ab207297; Abcam), and rabbit anti-GAPDH antibodies (1:1,000; cat. no. ab8245; Abcam) and were incubated at 4˚C overnight. The membranes were then incubated with the relevant goat anti-rabbit HRP-conjugated secondary antibodies (1:5,000; cat. no. ab7090; Abcam) at room temperature for 1 h, and the protein bands were visualized using ECL reagents (Amersham; Cytiva) and quantified by densitometry (ImageJ software; National Institutes of Health; http://rsbweb.nih.gov ).
9
Yeast Oxidative Stress Assay
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ROS was measured as described in our previous study [20 (link)]. In brief, the BY4741 yeast strain in glucose liquid medium treated with 0, 1, and 3 µM GENI or 10 µM RES was cultured with shaking at 28 °C and 160 rpm for 12 h. Each group was washed with PBS, added with 2′,7′-dichloro-dihydrofluorescein diacetate fluorescent probes to a final concentration at 10 µM, incubated for 1 h with shaking in the dark, and washed with PBS. The DCF fluorescence was determined using a microplate reader (BioTek, VT, USA) at 488 nm excitation and 525 nm emission wavelengths.
The MDA was measured as described in our previous study [20 (link)]. In brief, the BY4741 yeast strain in glucose liquid medium treated with 0, 1, and 3 µM GENI or 10 µM RES was cultured with shaking at 28 °C and 160 rpm for 12 h, washed with PBS, sonicated on ice for 5 min for protein extraction, and centrifuged at 4 °C and 12,000 rpm for 10 min to obtain the supernatant as protein samples. The protein concentration was determined using the BCA kit (CoWin Biotech, Beijing, China). According to the MDA kit (Nanjing Jiancheng Bioengineering, Nanjing, China) method, the MDA content of each group was determined. Detailed steps are shown in theSupporting Information (S2.1) .
The MDA was measured as described in our previous study [20 (link)]. In brief, the BY4741 yeast strain in glucose liquid medium treated with 0, 1, and 3 µM GENI or 10 µM RES was cultured with shaking at 28 °C and 160 rpm for 12 h, washed with PBS, sonicated on ice for 5 min for protein extraction, and centrifuged at 4 °C and 12,000 rpm for 10 min to obtain the supernatant as protein samples. The protein concentration was determined using the BCA kit (CoWin Biotech, Beijing, China). According to the MDA kit (Nanjing Jiancheng Bioengineering, Nanjing, China) method, the MDA content of each group was determined. Detailed steps are shown in the
10
Protein Extraction and Quantification
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In an ice bath, lung tissues were lysed with a tissue protein extraction kit (Pierce Chemical Co., Rockford, IL), USA) and a protease inhibitor (0.1 mM) was added. To determine the protein concentration, we used a BCA kit (Jiangsu Cowin Biotech Co., Ltd., Taizhou, China). With 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis, protein samples were separated and transferred onto nitrocellulose membranes (Luo et al. 2021 (link)). Immunoblotting was carried out with enhanced chemiluminescence (ECL) kit. Calculation of the band intensities was conducted using Gel Pro software (Media Cybernetics, Rockville, MD, USA).
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