Cells were lysed using RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with 1 mM phenylmethanesulfonyl fluoride (PMSF). After centrifugation at 14,000 rpm for 10 min at 4°C, a BCA protein assay kit (Beyotime, Shanghai, China) was used to determine the protein concentrations. Equal amounts of total proteins from each sample were loaded onto a 12.5% SDS-PAGE gel and transferred to PVDF membranes. The membranes were blocked and then incubated with primary antibodies at 4°C overnight. The primary antibodies used in this study were as follows: anti-NUF2 (1:800; 15731-1-AP, Proteintech Group, Inc, Wuhan, China), anti-ERBB3 (1:500; 10369-1-AP, Proteintech Group, Inc, Wuhan, China), anti-AKT (1:2,000; 10176-2-AP, Proteintech Group, Inc, Wuhan, China), anti-p-AKT (Ser473) (1:3,000; 28731-1-AP, Proteintech Group, Inc, Wuhan, China), anti-ERK1/2 (1:10,000; Abcam, Waltham, MA, USA), anti-p-ERK1/2 (Thr202/Tyr204) (1:1,000; #4695, Cell Signaling Technology, Boston, MA, USA) and anti-GAPDH (1:10,000; 10494-1-AP, Proteintech Group, Inc, Wuhan, China). The membranes were washed and incubated with the appropriate secondary antibodies. The target proteins bands on the membranes were detected using an ECL Western blotting Detection Kit (Beyotime, Shanghai, China). The gray values of the protein bands were analyzed using Quantity One software and normalized to GAPDH.
Ecl western blotting detection kit
Manufactured by Beyotime
Sourced in China
The ECL western blotting detection kit is a reagent system used for the detection of proteins in western blot analysis. It utilizes an enhanced chemiluminescent reaction to produce a luminescent signal that can be detected and quantified. The kit includes the necessary reagents for the chemiluminescent detection process.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Beyotime (4 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Cytation 3 cell imaging multi mode reader, by Agilent Technologies (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Ip lysis buffer, by Beyotime (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Bca protein assay kit, by Boster Bio (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Protein extraction kit, by Beyotime (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Anti β actin, by Proteintech (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Ab6671, by Abcam (1 mentions)
Anti actb antibody, by Sangon (1 mentions)
Ab16502, by Abcam (1 mentions)
Ab13556, by Abcam (1 mentions)
10 protocols using ecl western blotting detection kit
1
Western Blot Analysis of Cellular Proteins
2
Testicular Protein Extraction and Immunoblotting
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Following CO2 euthanization of mice, testes were removed, immediately snap-frozen, and stored at -80°C for further use. Protein extracts from mouse testes were prepared in IP lysis buffer (P0013, Beyotime, Shanghai, China) freshly supplemented with 1 mM PMSF (ST506, Beyotime). The samples were sonicated and centrifuged at 15,000 g for 1 hour at 4°C, the supernatant was collected, and the total protein concentration was determined by using the BCA Protein Assay Kit (P0012, Beyotime) via the Cytation 3 Cell Imaging Multi-Mode Reader (BioTek, Winooski, USA). Approximately 30-50 µg of protein from testis lysates collected at different ages of mice or time intervals after CdCl2 administration was separated by SDS-PAGE, and then transferred onto PVDF membrane (EMD Millipore, Bedford, USA) for immunoblotting with specific antibodies (Table 1
3
Cardiac Protein Expression Analysis
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The hearts were excised and the left ventricles were immediately frozen in liquid nitrogen before being stored at −80°C. Total protein from cardiomyocytes was extracted and the concentrations were determined by using the BCA protein assay kit (Boster Biotechnology, Wuhan, China). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a polyvinylidene difluoride (PVDF) membrane, blocked, and probed with primary antibodies against p-GSK-3β, t-GSK-3β, β-catenin, Bax, Bcl-2 and β-actin. The membrane was then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG. Finally, the bound antibody complexes were detected using the ECL Western blotting detection kit (Beyotime Institute of Biotechnology, Nanjing, China).
4
Western Blot Analysis of Protein Expression
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Protein was extracted from HCC cell lines using RIPA lysis buffer as per the protocol (Pierce Biotechnology Inc, Rockford, Illinois, USA). Protein concentration was determined via the bicinchoninic acid protein assay as per the Bio-Protocol (bio-protocol, Philadelphia, Pennsylvania, USA). An equivalent of 30μg of the protein extract was resolved via sodium dodecyl sulfate polyacrylamide gel electrophoresis and electro transferred (wet) to polyvinylidene difluoride membranes (EMD Millipore Corporation, Billerica, Massachusetts, USA). The membranes were initially blocked with 5% BSA in TBST (137mM NaCl, 20mM Tris HCl [pH 7.6], and 0.1% [v/v] Tween 20) for 1 h, followed by incubation overnight at 4°C with primary antibodies (1:1000) against AQP3, Erk, phospho-Erk, Akt, phospho-Akt, HIF-1α, LC3B, p62, cleaved caspase-3, and GAPDH/beta-tubulin (as loading controls) and horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000). Immunodetection was performed using an ECL Western Blotting Detection Kit (Beyotime, Shanghai, China). The relative protein expression levels were quantified by densitometric measurement of ECL reaction bands and normalized to GAPDH/beta-tubulin levels.
5
Protein Extraction and Western Blot Analysis
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Total proteins were extracted from fracture callus tissue or ATDC5 cells by using a Protein Extraction Kit (P0013, Beyotime, Shanghai, China) and the concentration was measured by a BCA Protein Assay Kit (P0012S, Beyotime, Shanghai, China). Western blotting was performed according to a previous study (Wang et al., 2019 (link)). Equal amounts of total protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred to polyvinylidene difluoride (PVDF) membranes. After being blocked with 5% skim milk solution and then incubated with diverse primary antibodies at 4 °C overnight, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse or goat-anti-rabbit antibodies (KPL, United States). Bands were visualized using ECL Western blotting Detection kit (P0018S, Beyotime, Shanghai, China) and analyzed using ImageJ software (NIH, United States). The list of primary and secondary antibodies is shown in Supplementary Table S2 .
6
Quantitative Western Blot Analysis
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Western blot was performed as previously described [30 (link)]. Primary antibodies for anti-MRP1 (1:2000, Abcom) and anti-β-actin (1:2000, proteintech) were used and protein bands were visualized using ECL Western Blotting Detection Kit (Beyotime Biotechnology, Shanghai, China). Band densitometry was calculated using Quantity One Software (Bio-Rad).
7
Western Blot Analysis of HAMP
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Cells and tissues were homogenized and lysed with ice-cold RIPA buffer supplemented with a proteasome and phosphatase inhibitor (#P0013B, Beyotime, China) for 30 min, and total proteins were extracted. The procedure for standard western blotting was performed as described in a previous study [23 (link)]. Primary antibodies anti-GAPDH (1:1000, #2118; CST) and anti-HAMP (1:1000, BM5068; Boster) were used to determine the expression of the corresponding proteins. Membranes were incubated in the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody at a 1:3000 ratio (Affinity Biosciences, Cincinnati, USA), and the protein bands were visualized by an ECL western blotting detection kit (Beyotime, Shanghai, China). GAPDH was used as the internal reference to normalize the protein loading. The images were quantified using ImageJ software.
8
Protein Isolation and Western Blot Analysis
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Total proteins from tissues and KFs were isolated by RIPA lysis buffer (Solarbio, Beijing, China) and the protein concentration was determined using BCA protein assay kit (Beyotime, Beijing, China). Equal amounts of proteins were subjected to 10% SDS-PAGE gel and transferred to PVDF membranes (Bio-Rad, Richmond, CA, USA). Then, after blocking with 5% non-fat dry milk, the membranes were incubated with primary antibodies proliferating cell nuclear antigen (PCNA), cleaved-caspase 3, collagen I and III and β-actin (CST, USA) at 4°C overnight. Subsequently, the membranes were incubated with HRP-conjugated secondary antibodies at room temperature for 2 h. Experiments were performed three times. Protein blots were visualized using an ECL western blotting detection kit (Beyotime).
9
Hippocampal Inflammatory Signaling Pathway
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Expression of TNF-α, NF-κB p65, and TLR-4 was analyzed by Western blot. Eight rats per group were sacrificed by rapid decapitation at 48 h after surgery. The brains were removed, and the hippocampi were separated on ice. Part of each hippocampus was rapidly frozen in liquid nitrogen. The hippocampal samples were homogenized, and proteins were extracted by RIPA buffer. Protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). The proteins were then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using 12% acrylamide gels and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was then incubated with the following primary antibodies at 4°C overnight: anti-TLR4 antibody (1:500, ab 13556, Abcam), anti-NF-κB p65 antibody (1:2,000, ab16502, Abcam), anti-TNF-α antibody (1:500, ab6671, Abcam). Anti-ACTB antibody (1:2,000, Sangon Biotech) was used as an internal control. After incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:2,000, ZB02301, ZSGB-BIO) for 2 h at room temperature, the blotted protein bands were visualized by enhanced chemiluminescence (ECL) Western blotting detection kit (P0018FS, Beyotime).
10
Western Blot Protein Analysis Protocol
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Protein was extracted using RIPA lysis buffer (Pierce Biotechnology Inc, Rockford, Illinois, USA). Protein concentration was determined via the bicinchoninic acid protein assay as per the Bio-Protocol (bioprotocol, Philadelphia, Pennsylvania, USA). An equivalent of 30μg of the protein extract was resolved via sodium dodecyl sulfate polyacrylamide gel electrophoresis and electro transferred (wet) to polyvinylidene di uoride membranes (EMD Millipore Corporation, Billerica, Massachusetts, USA) 32 (link) . The membranes were initially blocked with 5% BSA in TBST (137mM NaCl, 20mM Tris HCl [pH 7.6], and 0.1% [v/v] Tween 20) for 1 h, followed by incubation overnight at 4°C with primary antibodies (1:1000) against AQP3, Erk, p-Erk, Akt, p-Akt, p53, p-p53, p21, cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase 4 (CDK4), and GAPDH/beta-tubulin (as loading controls). The membranes were washed with PBS 3x each 5 min and then incubated with Horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000) at room temperature for 1 h. Immunodetection was performed using an ECL Western Blotting Detection Kit (Beyotime, Shanghai, China). The relative protein expression levels were quanti ed by densitometric measurement of ECL reaction bands and normalized to GAPDH/beta-tubulin levels 32 (link) .
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