Briefly, cells were lysed with a radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotech, China). Aliquots (30–50 µg) of proteins were resolved by SDS-PAGE (10%), then electro-transferred onto polyvinylidene difluoride membranes (Merck Millipore, USA) and immunoprobed. The protein-antibody complexes were detected using the ECL detection reagents (Beyotime Biotech, China). Primary antibodies used: anti-ZFP36L1 (ab42473), anti-HIF-1α (ab1) and anti-Hsp70 (ab5439) from Abcam (Cambridge, USA).
Ecl detection reagent
Manufactured by Beyotime
Sourced in China
The ECL detection reagent is a chemiluminescent substrate used for the detection of proteins in Western blot analysis. It emits light when exposed to horseradish peroxidase (HRP)-conjugated antibodies, allowing for the visualization of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (19 mentions)
Ripa lysis buffer, by Beyotime (12 mentions)
Bca protein assay kit, by Beyotime (10 mentions)
Ripa buffer, by Beyotime (7 mentions)
Protease inhibitor cocktail, by Roche (5 mentions)
Polyvinylidene difluoride membrane, by Merck Group (4 mentions)
Anti ha agarose, by Merck Group (3 mentions)
Anti β actin, by Santa Cruz Biotechnology (3 mentions)
Pvdf membrane, by Beyotime (3 mentions)
Opti mem, by Thermo Fisher Scientific (3 mentions)
Image lab software, by Bio-Rad (3 mentions)
Bca kit, by Beyotime (3 mentions)
Bca protein estimation kit, by Beyotime (3 mentions)
Bca method, by Thermo Fisher Scientific (2 mentions)
Nuclear and cytoplasmic protein extraction kit, by Beyotime (2 mentions)
Anti gclc, by Abcam (2 mentions)
Rabbit anti β actin, by Cell Signaling Technology (2 mentions)
Anti lamin b1, by Abcam (2 mentions)
Anti gclm, by Abcam (2 mentions)
Protein g agarose, by Thermo Fisher Scientific (2 mentions)
83 protocols using ecl detection reagent
1
Western Blot Analysis of Protein Expression
2
Investigating Protein Interactions in Cancer Cells
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Western blotting and immunoprecipitation were performed as previously reported [37 (link)]. Briefly, cells were lysed in MCLB, and clarified lysates were resolved by SDS-PAGE gel and transferred to poly-vinylidene difluoride membranes (Millipore, Billerica, MA, USA), then the membranes were blocked with 5% skimmed-milk powder in Tris-buffered saline with Tween-20 (TBS-T), incubated with the primary antibodies at 4 °C overnight, and then incubated with the secondary antibodies. The bands were detected by ECL detection reagents (Beyotime Biotechnology, Shanghai, China), and GAPDH was used as a loading control.
For immunoprecipitation, to investigate the interaction between DNMT1, DNMT3A, DNMT3B, and GRIA3 at the endogenous level, H460 and H1975 cells at 80–90% confluence were washed with ice-cold PBS three times before being lysed in IP lysis buffer. Then, the lysates were incubated with anti-DNMT1, anti-DNMT3A, or anti-DNMT3B antibodies separately overnight at 4 °C. Protein A/G-agarose beads were added for 2 h or overnight. The beads were collected and washed with lysis buffer for three times. The precipitated proteins were eluted and denatured in 2 × SDS loading buffer and analyzed by western blotting.
For immunoprecipitation, to investigate the interaction between DNMT1, DNMT3A, DNMT3B, and GRIA3 at the endogenous level, H460 and H1975 cells at 80–90% confluence were washed with ice-cold PBS three times before being lysed in IP lysis buffer. Then, the lysates were incubated with anti-DNMT1, anti-DNMT3A, or anti-DNMT3B antibodies separately overnight at 4 °C. Protein A/G-agarose beads were added for 2 h or overnight. The beads were collected and washed with lysis buffer for three times. The precipitated proteins were eluted and denatured in 2 × SDS loading buffer and analyzed by western blotting.
3
Spinal Cord Injury Protein Analysis
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Frozen spinal cord tissue samples (200 mg) isolated caudal to the injury were crushed in liquid nitrogen. Total protein extracts (100 ug/lane) were resolved using 10 or 15% SDS-PAGE and analyzed by western blot as described previously17 (link). All samples were incubated overnight at 4 °C with the following primary antibodies, rabbit anti-eIF5A1, 1:2000, Abcam; rabbit anti-RhoGDIα, 1:1000, Abcam; rabbit anti-β-actin, 1:5000, Abcam. Horseradish peroxidase-coupled secondary antibodies (1:5000; Abcam) were incubated in TBST for 1 h at room temperature. All samples were visualized using ECL detection reagents (Beyotime, China). Quantitative densitometric analysis was done using Image J software.
4
Western Blot Analysis of ER Stress Markers
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Cells were harvested and lysed in RIPA buffer containing a protease inhibitor cocktail (1:100). Total protein was extracted, and the concentration was determined by the BCA method (Thermo Fisher Scientific, USA). Then, equal amounts of total protein were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). The membranes were blocked and incubated with primary antibodies against FGF2 (36769, SAB), ATF6 (32008, SAB), GRP78 (3177S, CST), GRP94 (20292, CST), and GAPDH (5174, CST). Protein bands were then incubated with secondary anti-mouse or anti-rabbit peroxidase-linked antibodies (Beyotime). We then used enhanced chemiluminescent (ECL) detection reagents (Beyotime) to obtain the final result. Three replicates were performed for all WB analyses. WB results were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
5
Western Blot Analysis of Fibrosis Markers
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Western blot was performed as previously described (Ahn et al. 2019 (link)). The membranes were incubated with the primary antibodies: rabbit anti-TGF-β1 (1:1000, Proteintech, USA), rabbit anti-α-smooth muscle actin (α-SMA) (1:2000, ImmunoWay Biotechnology, USA), rabbit anti-collagen I (1:2000, Abcam, UK), rabbit anti-Smad3 (1:1500, Abcam, UK), rabbit anti-p-Smad3 (1:1500, Abcam, UK) or rabbit anti-β-actin (1:1500, Beyotime Institute of Biotechnology, China) overnight at 4 °C. The membranes were processed with the horseradish peroxidase-labelled secondary antibody (1:2000, Beyotime Institute of Biotechnology, China). The bands were visualised using the ECL detection reagents (Beyotime Institute of Biotechnology, China). The ratio of the density of bands of the detected protein to that of β-actin was used for statistical analysis.
6
Western Blotting Protocol for RPMS1 Detection
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Western blotting was performed as described previously [30 (link)]. Briefly, cells were lysed in mammalian cell lysis buffer, and proteins within the clarified lysates were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes for immunoblotting against the corresponding antibody. The results were revealed using enhanced chemiluminescent (ECL) detection reagents (Beyotime Co., Shanghai, China). The rabbit polyclonal anti-RPMS1 antibody was from Proteintech Group Inc. (Wuhan, Hubei, China), and the human anti-β-actin antibody was from Sigma-Aldrich Co. (St. Louis, MO, USA). A horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody was used as the secondary antibody (Promega, Madison, WI, USA).
7
Ginsenoside Rc Regulates Oxidative Stress
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Ginsenoside Rc was purchased from Yuanye Bio-technology Co., Ltd (Lot number: M27GB141849, purity > 98%). Isoproterenol and ML385 (Nrf2 inhibitor) were bought from Sigma Co. (St. Louis, MO, USA). Carboxymethyl cellulose sodium salt (CMC-Na) was from Shanghai Jinshan Chemical Co., Ltd (Shanghai, China). Creatine kinase (CK-MB, Lot number: 20210518), malondialdehyde (MDA, Lot number: 20210621) and glutathione (GSH, Lot number: 20210624) biochemical assay kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). TNF-α (Lot number: M21015903) and IL-1β (Lot number: V21013165) ELISA kits were from Wuhan Huamei Biotech Co., Ltd (Wuhan, China). Troponin T ELISA kit (Lot number: 20210618) was purchased from Solarbio Science & Technology Co., Ltd (Beijing, China). Nuclear and cytoplasmic protein extraction kit, BCA protein kit and ECL detection reagents were from Beyotime Institute of Biotechnology (Shanghai, China). Other reagents were of commercially analytical grade.
8
Protein Expression Analysis in Rat Heart
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The nuclear and cytoplasmic protein extracts were prepared using nuclear and cytoplasmic protein extraction kit (heart tissues of three rats in each group). Proteins were loaded (60 μg) and subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred to polyvinylidene fluoride membranes (Millipore Corporation) for 1.5 h at 100 V. Blocked with 5% nonfat milk for 1 h, the membranes were incubated with the primary antibodies: rabbit anti-Nrf2 (1 : 1500, Abcam), anti-GCLC (1 : 1500, Abcam), anti-GCLM (1 : 1000, Abcam), anti-Lamin B1 (0.1 μg/ml, Abcam), or rabbit anti-β-Actin (1 : 1500; Cell Signaling) overnight at 4°C. The membranes were processed with the respective horseradish peroxidase-labeled secondary antibody (1 : 3000). Bands were visualized using the ECL detection reagents (Beyotime Institute of Biotechnology, Shanghai, China).
9
Western Blot Analysis of Protein-Protein Interactions
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Cells were lysed in RIPA buffer (50 mM Tris-HCl [pH 8.0], 5 mM EDTA, 150 mM NaCl, and 0.5% Nonidet P-40 and a protease and phosphatase inhibitor cocktail (Bimake, China)), and the clarified lysates were resolved by SDS-PAGE and transferred to PVDF membranes for Western blot using ECL detection reagents (Beyotime, China). The immunoblots were processed according to standard procedures using primary antibodies directed to FBW7 (Bethyl, A301-720A), BRD7 (Bethyl, A302-304A), Cyclin E (CST, 20808), GAPDH (CST, 2118), HA (CST, 3724), Flag (CST, 14793), Myc (CST, 2278), V5 (CST, 13202), RCC2 (CST, 5104), RAD51 (CST, 8875), ERα (Santa Cruz, SC-514857), tubulin (Santa Cruz, SC-166729), NFATc1 (Abiocode, R2315–1). For immunoprecipitation, the supernatants were first incubated with S-protein agarose (Novagen) overnight at 4 °C, and the precipitates were washed three times with NETN buffer. To detect endogenous interaction, the clarified supernatants were first incubated with anti-FBW7 or NFATc1 antibody for two h and then protein G-agaroses (Thermo Fisher, 10004D) overnight. After washed three times with NETN buffer, the samples were collected and analyzed by Western blot.
10
Protein expression profiling in adipose and liver
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Frozen tissues (adipose or part of the liver), peritoneal macrophages, and PMC were separately homogenized and lysed by lysis buffer, then proteins were detected by western blot for ABCA1 (ATP-binding cassette transporter A1), ApoA-I (apolipoprotein AI), Apo-B (apolipoprotein B), PPARγ (peroxisome proliferator-activated receptor γ), and LXRα (liver X receptor α). Briefly, the total protein content in lysates was measured by Coomassie brilliant blue assay (Beyotime, Shanghai, China). Equal amounts of protein was electrophoresed on 10% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% nonfat dry milk, subsequently incubated 48 h with primary antibodies at 4°C, and then incubated with secondary antibodies (Boster, Wuhan, China) for 2 h. The blots were visualized using ECL detection reagents (Beyotime). Primary antibodies to ABCA1 and Apo-B were purchased from Abcam (MA, USA); PPARγ antibody and LXRα antibody were purchased from Proteintech (IL, USA); and ApoA-I antibody was purchased from MyBioSource (CA, USA).
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