Total protein in the heart tissues was extracted according to the instructions of the protein extraction kits (Solaibao Technology, Co. Ltd., Beijing, China), and quantified using bicinchoninic acid (BCA) protein assay kits (SolarBio, Beijing, China). Equal amounts of protein were loaded and separated using 8–12% sodium sulfate dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto PVDF membranes, which were blocked in 5% skimmed milk at 25°C for 1 h on a shaking table. Membranes were incubated with the appropriate concentrations of the following antibodies (Proteintech Group, Inc., Wuhan, China) at 4°C overnight: anti-Bcl-2 (1:2,000, Cat. No. 12789), anti-Bax (1:4,000, Cat. No. 50599), anti-Caspase-3 (1:2,000, Cat. No. 19677), anti-TGF-β (1:1,000, Cat. No. 21898, anti-SMAD2 (1:6,000, Cat. No. 12570), anti-SMAD3 (1:1,000, Cat. No. 25494), anti-SMAD4 (1:1,000, Cat. No. 10231), anti-SMAD7 (1:2,000, Cat. No. 25840), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:40,000, Cat. No. 10494). After three rounds of washing with Tris-buffered saline plus Tween (TBST), the membranes were incubated with a secondary anti-rabbit antibody (1:10,000, cat. no. 21991) at room temperature (RT) for 1 h. Finally, the immunoreactive bands were visualized using an enhanced chemiluminescence kit with a gel imaging system (Proteintech Group, Inc.).
Bca protein assay kit
Manufactured by Solarbio
Sourced in China, United States
The BCA protein assay kit is a colorimetric detection and quantification method for determining the total protein concentration in a sample. The kit uses bicinchoninic acid (BCA) to measure the reduction of copper ions by proteins in an alkaline environment, resulting in a purple-colored reaction that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (104 mentions)
Ripa lysis buffer, by Solarbio (65 mentions)
Ripa lysis buffer, by Beyotime (52 mentions)
Ripa buffer, by Solarbio (50 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (28 mentions)
Ripa buffer, by Beyotime (22 mentions)
Gapdh, by Proteintech (20 mentions)
Polyvinylidene difluoride membrane, by Merck Group (19 mentions)
Ab205718, by Abcam (17 mentions)
Polyvinylidene fluoride membrane, by Merck Group (17 mentions)
Gapdh, by Abcam (13 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (13 mentions)
Ipvh00010, by Merck Group (13 mentions)
Ripa buffer, by Thermo Fisher Scientific (13 mentions)
Bovine serum albumin (bsa), by Solarbio (12 mentions)
Total protein extraction kit, by Solarbio (11 mentions)
β actin, by Cell Signaling Technology (11 mentions)
Radioimmunoprecipitation assay (ripa), by Solarbio (10 mentions)
Quantity one software, by Bio-Rad (10 mentions)
Gapdh, by Cell Signaling Technology (10 mentions)
769 protocols using bca protein assay kit
1
Protein Expression in Heart Tissues
2
Metabolism and Immune Modulation of L. rhamnosus
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L. rhamnosus JL1 was previously isolated from infant feces and stored at −80 °C until use. L. rhamnosus JL1 was cultured at 37 °C for 18 h in Man Rogosa Sharp medium (Hope Technology Co., Ltd., Qingdao, China). Kits for TC (total cholesterol), TG (triglyceride), LDL-C (low density lipid-cholesterol) and HDL-C (high density lipid-cholesterol) were purchased from Jiancheng Institute of Bioengineering (Nanjing, China). IL-1β, IL-6, TNF-α and IFN-γ enzyme-linked immunosorbent assay kits were purchased from Huijia Biological Technology, (Xiamen, China). BCA Protein Assay Kits were purchased from Solarbio Science and Technology Co., Ltd. (Beijing, China). Other reagents were obtained from Sino pharm Co., Ltd. (Beijing, China).
3
Protein Expression Profiling in Min6 Cells
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Total protein was collected from Min6 cells (5×104 cells per well) lysed with RIPA lysis buffer (Vazyme Beyotime), and protein concentrations determined using BCA™ Protein Assay Kits (Solarbio; Beijing, China). Aliquots of 40 μg of protein were loaded onto each lane, and the proteins separated by 10% SDS-PAGE and transferred to PVDF membranes. The membranes were incubated with 5% skim milk and immunoblotted with primary anti-p-PI3K, anti-PI3K, anti-p-AKT, anti-AKT, anti-cleaved caspase-3, anti-pro caspase-3 and anti-GAPDH (Abcam, Cambridge, UK) antibodies overnight at 4°C, following by incubation with the respective secondary antibodies (Abcam) for 1 h at room temperature. The protein bands were visualized using ECL Kits (BestBio, Shanghai, China) and quantified with Image J Software (Bio-Rad, Shanghai, China). Each experiment was performed three times, all on cells of the same passage number.
4
Whole Protein Lysate Preparation and Analysis
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To prepare the whole protein lysate, BMDMs were lysed in NP-40 buffer supplemented with 1 mM PMSF on ice for 15 min. After centrifugation at 12000 rpm at 4°C for 10 min, the supernatant was carefully collected, and the protein concentration was determined by BCA Protein Assay Kits (Beijing Solarbio Science & Technology, Beijing, China) according to the manufacturer’s instructions. Subsequently, the samples were separated by 8% SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% milk for 2 h at room temperature and incubated with different primary antibodies at 4°C overnight. The next day, membranes were incubated with the indicated secondary antibodies at room temperature for 1 h. Finally, the bands were visualized by using LI-COR Odyssey, and the signals were analyzed by using Image J software.
5
Western Blot Analysis of Cell Signaling Proteins
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CAFs were homogenized in RIPA buffer (R0010, Solarbio). The homogenate was centrifuged at 4°C for 15 min at 15,000 g and then the supernatant was collected. Protein concentration was quantified with BCA protein assay kits (PC0020, Solarbio). Proteins were separated by 10% SDS polyacrylamide gels, and then transferred onto PVDF membranes. The membranes were blocked with 5% skimmed milk for 1 hour and then immunoblotted with CD143‐specific antibody (Cat. #ab254222, dilution 1:1000, Abcam), GAS1‐specific antibody (Cat. #17903‐1‐AP, dilution 1:1000, Proteintech), Cyclin D1 (Cat. #2978T, dilution 1:1000, CST), c‐Myc (Cat. #5605S, dilution 1:1000, CST) and β‐catenin (Cat. #8480S, dilution 1:1000, CST) at 4°C overnight. Membranes were then incubated with secondary antibodies conjugated with HRP (dilution1:10000, Abcam) at room temperature for 1 hour and bands were visualized using an ECL kit (FD8000, Fdbio science, Hangzhou, Zhejiang, China). β‐actin, GAPDH, or lamin B was used as loading control.
6
Cytokine Profiling in Colon Tissue
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Firstly, we selected all colon sections near anus. As reported in the previous study,49 (link) colons were cut into small parts and PBS with protease inhibitor (Solarbio, China) was added following thawing and weighing, then homogenization was conducted on ice followed by centrifugation (5 min at 13,000 rpm). The supernatant was then stored at −80°C until analysis. Next, the supernatant was normalized using BCA protein assay kits (Solarbio, China) according to the manufacturer’s instructions. TNF-α, IL-6, IL-1β, IL-18, IL-22, IL-9, IL-10, IL-4, IL-5, IFN-γ and IL-17A were measured for cytokine profiling assay using the Th1/Th2/Th9/Th17/Th22/Treg Cytokine 17-Plex Mouse ProcartaPlex™ Panel (EPX170-26087-901, Thermo Fisher Scientific). Then the 17 mouse cytokine levels were determined using Bio-Plex 200 System (Bio-Rad Laboratories).
7
Epithelial-Mesenchymal Transition Signaling Protocol
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DMEM-F12 medium and foetal bovine serum (FBS) were purchased from the Gibco (Invitrogen, USA). Penicillin, streptomycin and BCA protein assay kits were purchased from the Solarbio (Beijing, China). Polystyrene plate for transwell was purchased from the Corning (New York, USA). The primary antibodies were diluted 1:2000 before use, including OLA1 (Cat. #ab229090, Abcam), GAPDH (Cat. #ab9485, Abcam), GAPDH (Cat. #ab8245, Abcam), N-cadherin (Cat. #ab18203, Abcam), E-cadherin (Cat. #ab76055, Abcam), Snail (Cat. #PA5–11923, Invitrogen), Snail + Slug (Cat. # ab180714, Abcam), Zeb-1 (Cat. # ab155249 Abcam) and SMAD2 (Cat. #5339, Cell Signaling Technology), p-SMAD2 (Cat. #18338, Cell Signaling Technology), SMAD4 (Cat. #9515P, Cell Signaling Technology). TGF-beta 1 antibody (Cat. # 21898–1-AP) was purchased from ProteinTech (Wuhan, China). All the chemical compounds were analytically pure reagents.
8
Western Blot Analysis of Tight Junction Proteins
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Colon tissue was weighed and homogenized in RIPA extraction buffer (Solarbio, China). The homogenate was centrifuged at 4°C for 15 min at 15,000 g, then the supernatant was collected. The protein concentration was quantified with BCA protein assay kits (Solarbio, China) according to the manufacturer’s instructions. Proteins were separated by 10% SDS polyacrylamide gel, and then transferred onto PVDF membranes. The membranes were blocked with 5% skimmed milk for 1 h and then immunoblotted with primary antibodies against occuludin (ab167161, Abcam), claudin (36–4800, Thermo Fisher Scientific), helios (#89270, CST) at 4°C overnight. Membranes were then incubated with second antibodies labeled with HRP at room temperature for 1 h and bands were visualized using an ECL kits (Fdbio science, China). β-actin was used as a reference gene.
9
Protein Quantification and Western Blotting
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Lysis of the samples was performed in RIPA buffer containing 1 mM PMSF (Beyotime Technology, Shanghai, China) in an ice bath. BCA protein assay kits were used to determine the protein concentration in supernatants (Solarbio, Beijing, China). As in our previous study (X. Wang et al., 2020 (link)), an equal amount of protein from each sample was subjected to 10% SDS-PAGE and transferred to PVDF membrane. Primary antibodies (1:500 dilution) were incubated with the membranes at 4 °C overnight. Next, the membranes were incubated with secondary antibodies (1:1000 dilution) at room temperature for 1 h. An ECL kit (Solarbio Co. Ltd., Beijing, China) and a gel documentation system (Gel Doc EZ, BIO-RAD, United States) were used for protein band imaging. Protein band intensities were measured using ImageJ, with levels of ß-actin used to normalize the data.
10
Fenofibrate Improves Lipid Metabolism
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THP was obtained from Institute of Chinese Materia Medica, Chinese Academy of Medical Sciences (Beijing, China). Fenofibrate was purchased from Sigma-Aldrich (St. Louis, MO, USA). The common diet was provided by Zhejiang Medical College (Hangzhou, China). Commercial kits for the detections of TG and TC were obtained from Abbott Laboratories (Chicago, USA) and HDL-c and LDL-c were purchased from Biosino Biotechnology and Science (Beijing, China). Enzyme-linked immunosorbent assay (ELISA) kits for the quantitation of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and CYP7A1 were obtained from Meibiao Bioscience Co., Ltd. (Yancheng, China). Trizol reagents were purchased from ComWin Biotech (Beijing, China). PrimeScript™ RT reagent kit and SYBR Green qPCR kit were purchased from Takara (Dalian, China). RIPA lysis buffer was obtained from Beyotime (Shanghai, China). BCA protein assay kits were purchased from Solarbio (Beijing, China). Antibodies used for Western blot analysis were anti-TLR4 (Proteintech, Chicago, IL, USA), anti-TRAF6 (Affinity Biosciences Inc., Amherst, NH), and β-actin (Huaan Biotechnology, Hangzhou, China) and secondary antibodies were obtained from Huaan Biotechnology (Hangzhou, China). All primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).
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