Cells or small extracellular vesicels (sEVs) were lysed in RIPA buffer (50 mM Tris‐HCl pH 7.5, 150 mM NaCl, 1% Triton X‐100) containing protease inhibitor cocktail (Topscience). Proteins were separated by 12% acrylamide/bisacrylamide gel electrophoresis, transferred to PVDF membranes (Millipore) and probed with indicated primary antibody followed by horseradish peroxidase (HRP)‐conjugated secondary antibody. Immunodetection was carried out with the super sensitive ECL luminescence reagent (MA0186‐2, meilunbio). The integrated density of blot strips was analyzed by ImageJ and Prism 6.0 software to characterize the relative protein level.
Ecl luminescence reagent
Manufactured by Meilun
Sourced in China
The ECL luminescence reagent is a laboratory product designed to detect and quantify proteins in western blot analyses. It contains a chemiluminescent substrate that reacts with the enzyme horseradish peroxidase (HRP) to produce a light signal, which can be captured and measured using a suitable imaging system.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (4 mentions)
Ripa buffer, by Beyotime (4 mentions)
Bovine serum albumin (bsa), by Solarbio (2 mentions)
Protease inhibitor cocktail, by Topscience (1 mentions)
Chemiluminescence and fluorescence imaging system, by Bio-Rad (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Ab201792, by Abcam (1 mentions)
Phosphatase inhibitor, by Beyotime (1 mentions)
Ba2913, by Boster Bio (1 mentions)
Non fat milk, by Sangon (1 mentions)
Af1216, by Beyotime (1 mentions)
A0208, by Beyotime (1 mentions)
Pvdf membrane, by Roche (1 mentions)
Bs12478, by Bioworld Technology (1 mentions)
Bs6007m, by Bioworld Technology (1 mentions)
Goat anti mouse igg, by Bioworld Technology (1 mentions)
Skimmed milk, by BD (1 mentions)
0.2 μm pvdf membrane, by Merck Group (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Flag tagged beads, by Merck Group (1 mentions)
13 protocols using ecl luminescence reagent
1
Western Blot Protein Quantification
2
Western Blot Protein Analysis Protocol
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Total protein was extracted by using RIPA lysis buffer (NCM Biotech) with protease inhibitor cocktail and phosphatase inhibitor after transfection for 72 hours. 16-30 μg protein was loaded and electrophoresed on 8-12% SDS-PAGE gels, then transferred to a PVDF membranes (Millipore, Bedford, MA, USA). After sealing in NcmBlot blocking buffer (NCM Biotech) for 15 min, the membrane was incubated within primary antibody overnight at 4 °C. Then a horseradish peroxidase-conjugated secondary antibody (1:2000, Cell Signaling Technology) was used. Target protein bands were visualized using super sensitive ECL luminescence reagent (Dalian Meilun Biotechnology Co, Ltd.) in a Chemiluminescence and Fluorescence Imaging System (Bio-Rad). The antibodies used in this study were listed in Table S2 .
3
Western Blot Analysis of Autophagy and Inflammasome Proteins
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Proteins were extracted using RIPA buffer containing protease inhibitors and phosphatase inhibitors (Beyotime, China). Samples were homogenized on ice, centrifuged for supernatant at 12,000 g for 15 min at 4°C and heated to 100°C for 5 min. Protein extracts resuspended in sample loading buffer were separated by electrophoresis through 12% polyacrylamide gels and transferred to PVDF membranes (Millipore, USA). After blocking with 5% non-fat milk (Sangon Biotech Shanghai Co.,Ltd., China), membranes were incubated with primary antibodies anti-LC3 (4108S, CST, USA; 1: 1,000 dilution), anti-Beclin 1 (3738, CST, USA; 1: 1,000 dilution), anti-NLRP3 (15101S, CST, USA; 1: 1,000 dilution), anti-NLRC4 (ab201792, abcam, UK; 1: 1,000 dilution), anti-GAPDH (BA2913, Boster, China; 1: 1,000 dilution), anti-Tubulin (AF1216, Beyotime, China; 1: 1,000 dilution) and anti-Histone H3 (ab194681, abcam, UK; 1: 1,000 dilution) overnight at 4°C. Membranes were then washed and incubated with the horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (A0208, Beyotime, China; 1: 3,000 dilution) for 1 h at room temperature. Proteins were visualized using ECL luminescence reagent (Meilunbio, China). The gray-scale values of the bands were determined by Image J launcher broken symmetry software program (National Institutes of Health, Bethesda, MD, USA).
4
SOCS1 Protein Detection by Western Blot
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The activity of SOCS1 was detected by western blot assay. Proteins were extracted using RIPA lysis buffer, and their concentrations were determined via the BCA assay. Protein extracts were separated using 12.5% SDS-polyacrylamide gel and then their were transferred to PVDF membranes (Roche). After blocking with 3% BSA solution for 2 h, the membranes were incubated with primary antibodies at 4 ºC overnight. The membranes were washed with tris-buffered saline containing 0.1% Tween-20 and incubated with secondary antibody for 2 h at room temperature. The immunoreactivity was visualized using ECL luminescence reagent (Meilunbio, Dalian, China).
5
Western Blot Analysis of Metabolic and Inflammatory Markers
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Western blot was performed as previously described [26] . Brie y, proteins from the right inferior basal temporal lobe and cultured BV2 cells were lysed using RIPA lysis buffer. Equal amounts of protein were loaded onto SDS-PAGE gels, and following electrophoresis, were transferred to PVDF membranes. The membranes were then incubated overnight at 4°C with primary antibodies: rabbit anti-HK2 (Abcam, ab209847, 1:1000), rabbit anti-PKM2 (CST, 4053, 1:1000), rabbit anti-iNOS (Proteintech, 18985-1-AP, 1:1000), rabbit anti-TNF-α (CST, 11948, 1:1000), hamster anti-IL-1β (CST, 12507, 1:1000), rabbit anti-HIF-1α (Abcam, ab179483, 1:500), and mouse anti-β-actin (Bioworld, BS6007M, 1:5000). The next day, the membranes were treated with HRP-conjugated secondary antibodies anti-rabbit IgG (CST, 7074, 1:3000) and goat anti-mouse IgG (Bioworld, BS12478, 1:5000) respectively for 1 h at room temperature. Bands were observed with the sensitive ECL luminescence reagent (Meilunbio). Band densities were quanti ed with the Image J software (NIH). β-Actin was used as an internal standard.
6
CTI-2 Protein Expression Analysis
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After being cultured for 24 h in 6-well plates, cells were subsequently incubated with specific concentrations (0 µM, 25 µM) of CTI-2 for 24 h. Subsequently, their total protein content was determined after protein extraction using RIPA lysis buffer (Solarbio Life Sciences, Beijing, China). Use the BCA protein quantification kit to quantify the protein concentration. Then perform SDS-PAGE gel electrophoresis and transfer with 0.2 μm PVDF membrane (Merck, Darmstadt, Germany). After performing the blocking process in 5% skimmed milk (BD, Franklin Lakes, NJ, USA) for 1 h, the primary antibodies and membranes were incubated overnight at 4 °C. Next, discard the primary antibody and cleanse the membrane 3 times with TBST, and then incubated with the secondary antibodies for 1 h at room temperature (RT). Afterward, discard the secondary antibodies and wash the membrane four times in TBST for 10 min. Visualize the resultant antigen-antibody complexes using super sensitive ECL luminescence reagent (Meilunbio, Dalian, China). Images were obtained using the Tanon 5200 (Tanon Science & Technology Co., Ltd., Shanghai, China) imaging system. To normalize the protein load, and GAPDH is regarded as an internal control. Original western blotting images are shown in Supplementary Figures S2–S10 .
7
Western Blotting and Immunoprecipitation Protocols
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Standard methods of Western blotting and immunoprecipitation were used. Briefly, cells were lysed using RIPA buffer (Beyotime, Shanghai, China) and the supernatant was resolved by SDS-PAGE after centrifugation and transferred to PVDF membranes for Western blotting. Then, the membranes incubated with ECL luminescence reagent (Meilun Biotechnology Co., Ltd, Dalian, China) and exposed using Tanon 5200 Chemiluminescence Imaging System (Tanon Technology Co., Ltd, Shanghai, China).
The glutathione S-transferase (GST) pull-down assays and immunoprecipitation experiments were performed as described before [21 (link)]. The supernatant was incubated FLAG-tagged beads (A2220; Sigma-Aldrich, St. Louis, MO, USA) or HA-tagged beads (11815016001; Roche, Mannheim, Germany) or Glutathione Beads (SA008005; Smart-Lifesciences Biotechnology Co., Ltd, Changzhou, China) for 4 h. The precipitates were then washed five times and analyzed by Western blotting.
The glutathione S-transferase (GST) pull-down assays and immunoprecipitation experiments were performed as described before [21 (link)]. The supernatant was incubated FLAG-tagged beads (A2220; Sigma-Aldrich, St. Louis, MO, USA) or HA-tagged beads (11815016001; Roche, Mannheim, Germany) or Glutathione Beads (SA008005; Smart-Lifesciences Biotechnology Co., Ltd, Changzhou, China) for 4 h. The precipitates were then washed five times and analyzed by Western blotting.
8
Western Blot Analysis of Domperidone Treated Cells
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Cells were treated with or without 40 µM domperidone for 24 h, and then were lysed using RIPA buffer. Bichinconinic acid (BCA) assay kit was applied to measure the protein sample quantification, and each sample was prepared for same amount depending on protein concentration. Following SDS-PAGE separation, the proteins were subsequently transferred onto PVDF membranes (Millipore Corp). Subsequently, 5% non-fat milk was used to block the PVDF membranes for 1 h at RT, followed by incubation primary antibodies overnight at 4℃. Then, HRP-conjugated secondary antibodies were incubated for 2 h at RT. Specific bands were visualized by ECL luminescence reagent (Meilunbio).
9
Western Blot Analysis of PTEN/PI3K/AKT Pathway
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The transfected HGC-27 and AGS cells were collected and lysed with RIPA buffer (Beyotime Institute of Biotechnology). Subsequently, 40 µg/lane of proteins were loaded and resolved using 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. Then, the membranes were blocked with 10% BSA (Beijing Solarbio Science & Technology Co., Ltd.) for 1 h at room temperature. Subsequently, the membranes were incubated with primary antibodies: PTEN (cat. no. ab267787), PI3K (cat. no. ab154598), AKT (cat. no. ab8805), p-AKT (cat. no. ab250676) and GAPDH (cat. no. ab9485; all 1:1,000 dilution; Abcam) overnight at 4°C. Subsequently, proteins were incubated with the goat anti-rabbit IgG H&L (HRP; 1:2,000 dilution; cat. no. ab6721; Abcam) for 1 h at room temperature. The membranes were washed with 0.05% Tween-20 (TBS/Tween) 3 times. Finally, the protein bands were then analyzed with super sensitive ECL luminescence reagent (Dalian Meilun Biology Technology Co., Ltd.). The proteins expression levels were detected using a chemi-luminescence detection system with Quantity One software v.3.0 (Sigma-Aldrich; Merck KGaA).
10
Ovarian Tissue Endothelin Signaling Pathway
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Western blot analysis was performed. First, ovarian tissue proteins were extracted using RIPAlysis buffer containing a protease inhibitor cocktail and PMSF. Proteins were diluted to equal concentration adopting the detection of protein content, using BCA Protein Quantitative kit. The extractive was mixed with 5 × SDS-PAGE sample buffer. Secondly, the samples ware separated by 12% SDS-PAGE and transferred to PVDF membrane, using PowerPac™ Universal Power Supply and Trans-Blot@ SD Cell. After blocking with 5% fat-free milk for 1.5 h, the target protein bands were incubated with antibodies as following, overnight, 4 °C: Anti-ET-1 antibody (1:2000 dilution) Anti-ETA antibody (1:1000 dilution), anti-ETBR antibody (1:1000 dilution) and GAPDH (1:5000 dilution), β-actin (1:20000 dilution). After washing with TBST (5 min, 3 times), the membranes were incubated with secondary antibody(Goat anti rabbit IgG (H + L) Horseradish Peroxidase (HRP), Goat anti mouse IgG(H + L)HRP, for1.5 h at room temperature. At last, after immersing into the ECL luminescence Reagent(Meilunbio, China), the immunoreactive proteins were examined chemiluminescence using Biomolecular imager.
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