The whole hearts were cut into small pieces, then lysed on ice with cell lysis buffer supplemented with protease inhibitor and PMSF for 30 min. Then, samples were centrifuged at 12,000 g at 4 °C for 15 min. The concentration of total protein was determined using BCA protein detection kit (Thermo Scientific, Waltham, MA, USA); 50 μg total protein was separated on 10% SDS-PAGE gel, and then transferred to 0.45 μm PVDF membrane (Amersham Hybond, GE Healthcare, München, Germany). The PVDF membrane was blocked with TBST (Tris buffered saline, 0.2% Tween) buffer and 5% milk at room temperature for 2 h, and then incubated at 4 °C overnight with primary antibodies: rabbit anti-TGF-β1 (1:1,000), rabbit anti-Smad2/3 (1:1,000), rabbit anti-p53 (1:1,000), rabbit anti-Bax (1:1,000), rabbit anti-Bcl-2 (1:1,000), rabbit anti-β-actin (1:5,000). After been washed with TBST buffer, the membrane was incubated with goat anti-rabbit secondary antibody at room temperature for 1 h. Subsequently, the membrane was washed with TBST buffer and tested with ECL Kit (ThermoFisher, USA). Protein expression of β-actin was used as an internal control. Image J software (National Institutes of Health, Bethesda, Maryland, USA) was used for gray-scale quantitative analysis.
Ecl kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, Germany, United Kingdom, Japan, Australia, Panama, Sweden, Israel
The ECL kit is a laboratory product designed for the detection and quantification of proteins in Western blot analysis. It provides a chemiluminescent substrate that reacts with the enzyme-labeled target proteins, generating a detectable light signal. The kit's core function is to enable the visualization and analysis of protein expression levels.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (224 mentions)
Bca kit, by Thermo Fisher Scientific (113 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (109 mentions)
Ripa lysis buffer, by Beyotime (99 mentions)
Ripa buffer, by Thermo Fisher Scientific (88 mentions)
Bca protein assay kit, by Beyotime (58 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (55 mentions)
Polyvinylidene difluoride membrane, by Merck Group (51 mentions)
Pvdf membrane, by Bio-Rad (47 mentions)
Bca kit, by Beyotime (45 mentions)
Ripa buffer, by Beyotime (43 mentions)
Pvdf membrane, by Thermo Fisher Scientific (37 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (37 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (36 mentions)
Protease inhibitor cocktail, by Merck Group (35 mentions)
Polyvinylidene fluoride membrane, by Merck Group (34 mentions)
Protease inhibitor cocktail, by Roche (32 mentions)
Gapdh, by Cell Signaling Technology (32 mentions)
Nitrocellulose membrane, by Bio-Rad (26 mentions)
Gapdh, by Abcam (25 mentions)
865 protocols using ecl kit
1
Western Blot Analysis of Cardiac Proteins
2
Western Blot Analysis of Protein Expression
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Proteins were extracted as previously described [19 (link)]. Subsequently, SDS-PAGE (7.5–10% polyacrylamide gels) was used to separate the proteins, which were then transferred to polyvinylidene fluoride (PVDF) membranes (Thermo Scientific). The membranes were blocked with 5% skim milk and then incubated with the primary antibodies (1:1000; CST, Danvers, MA, USA) overnight, followed by incubation with secondary antibodies (1:2000; CST, Danvers, MA, USA) at room temperature. The immunoreactive protein bands were visualized using an enhanced chemiluminescence (ECL) kit (Thermo Scientific). A Luminescent Image Analyzer LAS4000 (Fuji Film, Tokyo, Japan) and ImageJ software (NIH, Bethesda, MD, USA) were used to detect and quantify the protein band signals. U0126 ERK pathway inhibitor was obtained from Sigma-Aldrich (Danvers, MA, USA). ML385 NRF2 pathway inhibitor was purchased from MedChemExpress (Shanghai, China).
3
Protein Extraction and Western Blot Analysis
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Both types of cells were lysed on ice with M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, USA) supplemented with 10% protease inhibitor cocktail (Roche, Mannheim, Germany) to isolate total protein and then measured by a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, USA) in accordance with the manufacturer’s instructions. Protein samples supplemented with loading buffer in equal proportions were electrophoresed on NuPAGE™ Bis-Tris Protein Gels (Invitrogen, Waltham, MA, USA) and then transferred onto polyvinylidene fluoride (PVDF) membranes. After incubation with 5% skim milk (5% w/v) for 2 h at room temperature, the membranes were co-incubated overnight at 4 °C with the following primary antibodies specific for GAPDH (1:1000; Cell Signaling Technology, USA), Runx2 (1:1000; Cell Signaling Technology, Danvers, MA, USA), Osx (1:1000; Abcam, Cambridge, UK), Ocn (1:2000; Abcam, UK), ELK4 (1:1000; Proteintech, Rosemont, IL, USA), GM130 (1:1000; Proteintech, USA), CD9 (1:1000; Proteintech, USA), and TSG101 (1:1000; Proteintech, USA). Next, the peroxidase-conjugated secondary antibody (1:5000; ZS-GB-BIO, Beijing, China) was utilized to treat the PVDF membranes for 2 h at room temperature. The ECL kit (Thermo Fisher Scientific, USA) was employed to visualize the signals. Densitometry analysis was conducted with ImageJ software.
4
Western Blot Analysis of Cell Signaling Proteins
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Protein sample preparation and western blotting were performed as previously described [43 (link)]. Protein samples were separated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membrane was blocked with milk and subsequently incubated with primary antibodies against glyceraldehyde 3-phosphate dehydrogenase (1:1000; Affinity, USA), PCNA (1:1000; CST. Inc., USA), cyclin B1 (1:1000; CST. Inc., USA), Akt antibody (1:1000; CST. Inc., USA), PI3K (1:1000; CST. Inc., USA), and anti-mTOR (1:1000; CST. Inc., USA). The membranes were then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10000; Affinity Bio., USA) and goat anti-mouse IgG (1:10000; Affinity Bio., USA) secondary antibodies. The results were analysed using an ECL kit (Thermo Fisher Scientific).
5
Biochemical Analysis of AKT Signaling
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LY294002, DAPI, antibodies against β-actin, α-tubulin (DM1A), OGT and HA were products of Sigma-Aldrich Corp. (St. Louis, MO, USA). Antibody against Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was the product of Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA). Antibodies against AKT, AKT-pS473, AKT-pT308, Bad, p-Bad (Ser136), Cleaved Caspase3, Caspase3, cleaved PARP were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). ECL kit, protein G agarose beads, and antibody against O-GlcNAc-modified proteins (RL2) were purchased from Thermo Fisher Scientific (Rockland, IL, USA). Antibody against OGA was a gift from S. W. Whiteheart (University of Kentucky College of Medicine, Lexington, KY). Peroxidase-conjugated anti-mouse and anti-rabbit IgG were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Alexa 488- conjugated goat anti-mouse IgG, and TO-PRO-3 iodide (642/661) were from life Technologies (Grand Island, NY, USA). CellTiter 96® Non-Radioactive Cell Proliferation Assay and DeadEnd™ Fluorometric TUNEL kits were from Promega (Madison, WI, USA). Caspase 3 Assay Kit (Colorimetric) was from Abcam (Cambridge, MA, USA).
6
Protein Extraction and Western Blot Analysis
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Firstly, RIPA solution (Beyotime, Shanghai, China) was used to extract total proteins from mouse brain tissue or cells. Total protein concentration was measured and quantified using a BCA kit (Thermo Fisher Scientific, Grand Island, NY, USA). Protein samples mixed with 5×loading buffer were denatured in boiling water, separated by gel electrophoresis and transferred to PVDF membrane (Merck Group, Darmstadt, Germany). Closed with 5% skim milk for 1h and incubated with the following primary antibodies at 4° C overnight: rabbit monoclonal anti-Nampt (1:1000; 11776-1-AP; Proteintech, China), β-actin (1:1000; ab241153; Abcam), rabbit monoclonal anti-METTL3 (1:1000; 15073-1-AP; Proteintech). PVDF membrane is then paired with corresponding HRP labeled anti-Rabbit IgG (1:4000; #7074S; CST) was incubated at room temperature for 2h. Strip visualization was done using the ECL kit (Thermo Fisher Scientific, USA). The results were quantified using software ImageJ.
7
Western Blot Analysis of NSCLC Cell Signaling
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NSCLC cells were lysed in RIPA lysis buffer (KeyGEN, Nanjing, China), and the concentration of protein was detected by BCA Assay kit (Solar life science, Beijing, China). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 10%) was performed to separate the equal amounts of protein (30 μg), and proteins were then shifted onto polyvinylidene difluoride membrane (PVDF, Thermo Fisher Scientific). Five percent nonfat dried milk in TBST was applied to block the PVDF membrane for 1 h. PVDF membrane was incubated at 4°C overnight with the primary antibodies: E-cadherin (Abcam, Cambridge, MA, USA, 1:1000), N-cadherin (Abcam, 1:1000), p-Akt (Abcam, 1:1000), Akt (Abcam, 1:1,000), p-ERK (Abcam, 1:1000), ERK (Abcam, 1:1000), cleaved caspase 3 (Abcam, 1:1000), p27 Kip1 (Abcam, 1:1000), cyclin D1 (Abcam, 1:1000), CDK2 (Abcam, 1:1000) and β-actin (Abcam, 1:1000). Then, the membranes were incubated with HRP-conjugated secondary antibodies (Abcam; 1:5000) for 1 h. Protein bands were visualized using the ECL kit (Thermo Fisher Scientific). β-actin was used as the loading control. IPP 6.0 (Image-Pro Plus 6.0) was used for the densitometry analysis.
8
Western Blot Analysis of Protein Expression
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The protein samples from the cells were extracted using the cell lysis buffer (cat. no. P0013; Beyotime Biotechnology Inc.) and the protein content was measured using a BCA Protein Assay Kit (cat. no. P0012S; Beyotime Biotechnology Inc.). The protein samples (40 µg) were subjected to 15% SDS-PAGE for separation and transferred onto nitrocellulose membranes. After blocking in fresh 5% non-fat milk at room temperature for 2 h, the membranes were incubated overnight at 4˚C with primary antibodies, followed by incubation with a HRP-conjugated secondary antibody at room temperature for 2 h. An enhanced chemiluminescence (ECL) kit (Thermo Fisher Scientific, Inc.) was used to visualize the signals. The antibodies (Affinity Biosciences) used included: Anti-SIRT1 (cat. no. DF6033; 1:1,000), anti-phosphorylated (p-)-AMPK (cat. no. AF3423; 1:1,000), anti-AMPK (cat. no. AF6423; 1:1,000), anti-Cox-2 (cat. no. AF7003; 1:1,000), anti-iNOS (cat. no. AF0199; 1:1,000), anti-Bcl-2 (cat. no. AF6139; 1:1,000), anti-Bax (cat. no. AF0120; 1:1,000), anti-cleaved-caspase-3 (cat. no. AF7022; 1:1,000), anti-cleaved-caspase-9 (cat. no. AF5240; 1:1,000), anti-GAPDH (cat. no. AF7021; 1:5,000) and the goat anti-rabbit IgG secondary antibody (cat. no. S0001; 1:5,000). Protein expression levels were semi-quantified using Image-Pro Plus software version 6.0 (Media Cybernetics, Inc.).
9
Western Blot Protein Isolation
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Total protein was isolated by RIPA lysis buffer (Thermo Fisher Scientific). Protein samples were separated by 10% SDS-PAGE and transferred to a PVDF membrane. Thereafter, the membranes were incubated with primary antibodies at 4°C overnight. Blots were detected using an ECL kit (Thermo Fisher Scientific) [25 ].
10
Protein Expression Analysis via Western Blot
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Cells were disrupted by lysis buffer and protein content was determined using the BCA Protein Assay Kit. The quantified protein samples were then separated by SDS-PAGE and transferred to PVDF membranes, followed by sealing with 5% skim milk for 1 hour at room temperature. The PVDF membranes carrying the samples were incubated with primary antibodies overnight at 4°C, and then with secondary antibody for 2 h at room temperature. The signal was displayed with an enhanced chemiluminescence (ECL) kit (Thermo Fisher Scientific, Inc.). And, the protein expression levels were semi-quantified using Image-Pro Plus version 6.0 (Media Cybernetics, Inc.) software. The antibodies used in this study were purchased from Abcam and were used at the following concentrations: anti-Bcl-2 (1:1000), anti-Bax (1:1000), anti-cleaved caspase3 (1:100), anti-caspase3 (1:500), anti-PDE3B (1:2000), anti-p-AMPK (1:1000), anti-AMPK (1:1000), anti-p-mTOR (1:1000), anti-mTOR (1:10,000), anti-GAPDH (1:2500), and goat anti-rabbit IgG H&L (HRP) (1:2000).
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