Cell lysates subjected to SDS polyacrylamide electrophoresis on pre-cast 4–20% gradient gels (GenScript) and separated proteins transferred to PVDF immunoblotting membrane (Bio-Rad). PVDF membrane was blocked in 5% w/v skim milk (from powder, EMD Millipore), and stained with primary antibodies and secondary horseradish peroxidase-conjugated antibody (see Antibodies, above). Signal was developed with ECL western blotting substrate (Pierce Chemical) and captured on x-ray film. For re-probing with additional antibodies, blots were stripped (Re-Blot Plus, Millipore) and blocked again with skim milk, as above.
Ecl western blotting substrate
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, France, Germany, United Kingdom, Japan, Belgium, Italy
ECL Western Blotting Substrate is a chemiluminescent detection reagent used in western blotting applications to visualize and analyze target proteins. It produces a luminescent signal upon reaction with a horseradish peroxidase (HRP) enzyme label.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (51 mentions)
Pvdf membrane, by Merck Group (42 mentions)
Ripa buffer, by Thermo Fisher Scientific (28 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (25 mentions)
Ripa buffer, by Merck Group (25 mentions)
Protease inhibitor cocktail, by Roche (21 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (19 mentions)
Nitrocellulose membrane, by Bio-Rad (18 mentions)
Chemidoc xrs system, by Bio-Rad (17 mentions)
Pvdf membrane, by Bio-Rad (14 mentions)
Bca kit, by Thermo Fisher Scientific (13 mentions)
Quick start bradford protein assay, by Bio-Rad (12 mentions)
β actin, by Merck Group (12 mentions)
Phosphatase inhibitor cocktail, by Roche (11 mentions)
Bca protein assay kit, by Beyotime (10 mentions)
Bca assay, by Thermo Fisher Scientific (10 mentions)
Pvdf membrane, by Thermo Fisher Scientific (10 mentions)
Nitrocellulose membrane, by GE Healthcare (10 mentions)
Protease inhibitor cocktail, by Merck Group (9 mentions)
Horseradish peroxidase conjugated anti rabbit or mouse igg, by Vector Laboratories (9 mentions)
491 protocols using ecl western blotting substrate
1
Protein Separation and Western Blotting
2
Western Blot Protocol for Protein Analysis
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Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitor cocktail (Sigma-Aldrich), sonicated, and clarified by centrifugation. Equal amounts of protein were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose membranes. Membranes were incubated with primary antibodies and secondary antibodies conjugated to horseradish peroxidase (KPL, Gaithersburg, MD, USA). Immunoreactive bands were visualized using an ECL western blotting substrate (Pierce Chemical, Rockford, IL, USA). Blots were scanned using FluorChemFC2 software with the AlphaImager system (Alpha Innotech Corp., San Leandro, CA, USA). Figures shown are representative of three or more experiments each.
3
Western Blot Protein Analysis
4
Western Blot Analysis of Apoptosis and Oxidative Stress Markers
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HK-2 cells were thoroughly lysed using RIPA lysis buffer. Total protein in the lysate was assessed using Bradford assay. Equal amounts of proteins were loaded onto sodium dodecyl sulphate polyacrylamide gel. The separated samples were then transferred onto a PVDF membrane. The membrane was further blocked using 5% skim milk and incubated with primary antibodies (MCL-1, Abcam, ab32087, 1:1000), PCNA (Abcam, ab29, 1:1500), Cyclin D1 (Abcam, ab16663, 1:1000), BAX (Abcam, ab32503, 1:1000), Bcl-2 (Abcam, ab182858, 1:1000), Cleaved-caspase-3 (Cell signaling, #9664, 1:1500), NOX1 (Abcam, ab121009, 1:1000), NOX2 (Abcam, ab310337, 1:1500), SOD1 (Abcam, ab51254, 1:1000), SOD2 (Abcam, ab68155, 1:1000), Caspase-3 (Abcam, ab184787, 1:1000), β-Actin (Abcam, ab8226, 1:3000), and GAPDH (Abcam, ab8245, 1:2000), overnight at 4 °C. Further, the blots were washed and incubated with secondary antibody for 1 h at RT. Finally, the bands were visualized using ECL western blotting substrate (Invitrogen, 32109, USA). Western blot images were quantified by using ImageJ (V1.8.0.112, National Institutes of Health, Bethesda, MD), with the density of each band normalized to that of β-Actin or GAPDH (Additional file 2 ).
5
Western Blot Analysis of Metabolic Enzymes
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Cultured cells were lysed in RIPA buffer (Boster, Wuhan, China) with 1% Phenylmethanesulfonyl fluoride (Beyotime, China) following the manufacturer’s instruction. Protein was loaded and seperated by SDS-PAGE gel and transferred onto PVDF membrane (Bio-Red Laboratories Inc, USA). Then, the polyvinylidene difluoride (PVDF) membrane were incubated with anti-ACAA2 (1:1000, Abcam, Cambridge, MA, USA), anti-HSD17B12 (1:1000, Abcam) or anti-actin (1:1000, Abcam) at 4 °C overnight. After washing 3 times by Tris Buffered Saline with Tween 20 (TBST), the PVDF membrane was incubated with goat anti-rabbit IgG (1:3000, Abcam) for 1 h at room temperature. Finally, protein bandings were obtained by enhanced chemiluminescence (ECL) Western Blotting Substrate (Invitrogen USA). And the signal intensities were captured by a Tanon 5200 chemiluminescence/fluorescence image analysis system. β-actin was used as the endogenous control.
6
Comprehensive Protein Extraction and Analysis
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Cells were lysed in RIPA buffer [25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1% NP-40] supplemented with 1× protease and phosphatase inhibitor (cat. no. 78441; Thermo Fisher Scientific). After centrifugation at 4 °C for 20 min, the supernatant was carefully collected and quantitated, before being stored at −80 °C for further analysis. Nuclear protein was isolated using the Nuclear Extraction Kit (cat. no. ab113474; Abcam). Equal amounts of proteins and exosomes were loaded onto 7.5 or 12.5% SDS polyacrylamide gel, and then transferred to a nitrocellulose membrane (cat. no. 88025; Thermo Fisher Scientific). The membrane was blocked for 1 h in 5% nonfat milk or bovine serum albumin, and then incubation with the indicated primary antibodies took place over-night at 4 °C. After incubation with HRP-conjugated goat anti-rabbit or goat anti-mouse IgG (cat. no. G-21234 and G-21040; Invitrogen) for 1 h at room temperature, the signals were detected using ECL Western Blotting Substrate (cat. no. 32109; Pierce/Thermo Fisher Scientific). To remove the primary antibodies for reblotting with the other primary antibody, Stripping Buffer for Western blot (Thermo Fisher Scientific) was applied to some blots. The specific bands were quantitated using Image J (Version 1.51s; National Institute of Health, Rockville, MD, USA).
7
Gene Expression and Apoptosis Evaluation
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Total RNA was isolated using Trizol reagent and cDNAs were synthesized by a RT-PCR Kit (Takara, Japan). Specific primers were designed for RT-PCR and qPCR reaction (Table 1 ). The conditions and procedures were referred to the instructions. The proteins were extracted with RIPA buffer (Boster, China) and the BCA Protein Assay Kit (Boster, China) was used to detect protein concentration referring to the instructions. Proteins were isolated by SDS-PAGE and transferred to PVDF membrane. The membrane was blocked with 5% skim milk powder and then incubated with rabbit anti-ITPKB antibody, 1:200 ((Bioss, China), rabbit anti-Caspase-3 antibody, 1:300 (Bioss, China), rabbit anti-Bcl-2 antibody, 1:300 (Bioss, China) and mouse anti-β-actin antibody, 1:6000 (Bioss, China). The proteins bandings were detected with ECL Western Blotting Substrate (Invitrogen, USA).
8
Western Blot Protein Analysis
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The proteins were extracted from cells using RIPA lysis buffer (Beyotime, Shanghai, China), followed by quantification using a BCA Protein Assay Kit (Abcam, UK). Then proteins were undergone SDS-PAGE electrophoresis and then transferred to PVDF membranes, followed by sealing with 5% skim milk. Subsequently, the membranes were hatched with primary antibodies at 4 °C overnight and then incubated with horseradish peroxidase-conjugated secondary antibodies. Immunoblots were detected using ECL western blotting substrate (Invitrogen, USA). The experiment was in triplicate.
9
Immunoblot Analysis of Autophagy and Hippo Pathway
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Total protein was extracted with RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific). The protein concentration was determined using BCA Protein Assay Kit (Abcam, Cambridge, UK). Then the protein extracts were separated by SDS-PAGE and transferred onto a PVDF membrane (Millipore, Billerica, MA, USA). Membranes were blocked with 5% skim milk and incubated with primary antibodies against p62 (Abcam, 1/10000-1/50000), LAMP1 (Abcam, 1/1000-1/10000), LC3 (Abcam, 1/2000), TAZ (Abcam, 1/50-1/200), LATS1 (Abcam, 1/5000), LATS2 (Abcam, 1 µg/ml), CK1 (Abcam, 1 µg/ml), YAP (Abcam, 1/5000), p-YAP (Abcam, 1/10000), GAPDH (CST, 1/1000), HUR (Abcam, 1/1000) and β-actin (Abcam, 1 µg/ml) for 4 °C overnight. Then, the membranes were hatched with horseradish peroxidase-labeled secondary antibodies for 2 hours at room temperature. After being washed again, the protein detections were used ECL Western Blotting Substrate (Invitrogen). The experiment was conducted at least three times. Original western blots are included in Supplemental Material.
10
Protein Expression and Detection
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Total protein was extracted from in cells with RIPA lysis buffer (Thermo Fisher, USA). The concentration of protein was measured using a BCA protein assay reagent (Beyotime, Shanghai, China). Protein was separated by 10% SDS-PAGE and transferred onto PVDF membranes (Millipore, USA). The membranes were blocked with 5% defatted milk in TBST and incubated with primary antibody, including Anti-PKM (1/10000), Anti-ENO1 (1/1000) and Anti-GAPDH (1/500-1/10000) at 4°C overnight. Next, the membranes were washed and then incubated with horseradish peroxidase-labeled secondary antibody (1/10000) at room temperature for 2 h. Finally, the blot was visualized by using the ECL western blotting substrate (Invitrogen). The experiment was independently conducted in triplicate. The original images were provided as Supplementary file.
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