Cells were lysed in RIPA lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% sodium deoxycholate, 1% NP-40, 1 mM PMSF and 1 mM EDTA. Then, extracts were centrifuged at 24,750 × g at 4°C for 15 min to remove insoluble material. Total protein concentrations were determined using a bicinchoninic acid assay (Beyotime Institute of Biotechnology), according to the manufacturer's protocol. An equivalent quantity of total protein (40 µg) per well was diluted in sample buffer containing 100 mM dithiothreitol and heated to 98°C for 5 min. Lysates were separated using 10–15% SDS-PAGE (Bio-Rad Laboratories, Inc.) and subsequently transferred to PVDF membranes. The membranes were blocked in 5% non-fat dry milk for 2 h at room temperature and incubated overnight at 4°C using primary antibodies (1:500) against β-actin, Runx2, 4E/BP 1, p-4E/BP1, S6K1 and p-S6K1. Membranes were washed in TBS with Tween-20 (TBS-T), and incubated with horseradish peroxidase-conjugated secondary antibody (1:2,000) at room temperature for 1 h. Membranes were washed three times for 20 min in TBS-T, and protein bands were visualized by enhanced chemiluminescence (Proteintech Group, Inc.). Band intensities were measured and quantitated using Quantity One software (version 4.6.6; Bio-Rad Laboratories, Inc.) and β-actin was used for normalization.
Enhanced chemiluminescence
Manufactured by Proteintech
Sourced in United States
Enhanced chemiluminescence is a laboratory technique used to detect and quantify specific proteins in Western blot analysis. It utilizes a chemiluminescent substrate that emits light when it reacts with the enzyme-labeled target protein, allowing for visualization and analysis of the protein.
Automatically generated - may contain errors
Lab products found in correlation
Quantity one software, by Bio-Rad (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Sds page, by Bio-Rad (1 mentions)
Bicinchoninic acid assay, by Beyotime (1 mentions)
Vimentin, by Proteintech (1 mentions)
Nf κb2, by Proteintech (1 mentions)
Hsp70, by Boster Bio (1 mentions)
β actin, by Proteintech (1 mentions)
α sma, by Proteintech (1 mentions)
Anti tsg101, by GeneTex (1 mentions)
Bca protein assay, by Solarbio (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Proteintech (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Phosstop, by Roche (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Hrp conjugated affinipure goat anti rabbit igg h l, by Proteintech (1 mentions)
Hrp conjugated affinipure goat anti mouse igg h l, by Proteintech (1 mentions)
Rabbit anti calnexin, by Proteintech (1 mentions)
Rabbit anti cd9, by Proteintech (1 mentions)
Rabbit anti cd81, by Proteintech (1 mentions)
10 protocols using enhanced chemiluminescence
1
Protein Expression Analysis via Western Blot
2
Western Blot Analysis of Protein Expression
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Total protein was extracted with radioimmunoprecipitation assay (RIPA) lysis buffer. The cell lysate was then quantified and boiled for 5 min before 30 μg of the samples were separated by10% SDS-PAGE. Proteins were then electrotransferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA) and blocked for 2 h with 3% BSA at room temperature. Subsequently, the membranes were incubated overnight at 4°C with primary antibodies for the detection of NF-κB2 (1:1,000; Proteintech, USA), MMP2 (1:1,000; Proteintech), HSP70 (1:500; Boster, USA), CD9 (1:1,000; Proteintech), FAP-1 (1:1,000; Abbkine, USA), vimentin (1:1,000; Proteintech), α-SMA (1:1,000; Proteintech), and β-actin (1:3,000; Proteintech). The membranes were then washed (3 × 10 min) with Tris-buffered saline containing Tween-20 (TBST). After incubation for 90 min at room temperature with anti-rabbit secondary detection antibodies (1:5,000; Proteintech), immunoreactive bands were visualized by enhanced chemiluminescence (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
3
Protein Expression Analysis by Western Blot
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The harvested cells were lysed in RIPA buffer (50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.1% SDS, 0.5% deoxycholic acid, 1% Triton X-100) supplemented with 10% PMSF. Protein concentration of cell lysate was determined using BCA protein assay kit (Pierce). Equivalent amount of protein (50 ug) was fractionated by precast mini polyacrylamide gels (SurePAGE™, Bis-Tris, 4–20%) (GenScript, Nanjing, Jiangsu) and undergone western blotting. The proteins were visualized by enhanced chemiluminescence (Proteintech Group, Inc., Chicago, IL) according to the manufacturer’s instructions.
4
Western Blot Analysis of Extracellular Vesicle Markers
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Protein were prepared with a detergent buffer, and the protein concentration was determined using the bicinchoninic acid (BCA) protein assay (Solarbio, Beijin, China). Equal amounts of protein samples were separated by a 12% gel using sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The membranes were probed with anti-TSG101 (GeneTex, USA), anti-CD63 (Immunoway,USA) antibodies overnight at 4˚C. Immune complexes were detected by enhanced chemiluminescence (Proteintech, Chicago,USA).
5
Western Blot Analysis of Cell Signaling
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Cell lysates were harvested with RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) containing Protease Inhibitor Cocktail (Calbiochem, San Diego, CA, USA) and PhosSTOP (Roche Applied Science, Rockford, IL, USA). Protein concentrations in supernatant were detected by the bicinchoninic acid (BCA) assay (Beyotime Biotechnology). Proteins were separated by western blot and then transferred to polyvinylidene difluoride membranes (Merck Millipore), which were then blocked with Tris-buffered saline (TBS; Sangon Biotech, Shanghai, China) containing 0.1% Tween 20 with 5% (w/v) non-fat milk. Membranes were incubated overnight at 4 °C with the primary antibody and then incubated for 2 h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000, Proteintech). Antibody dilution ratios were as follows: TLR2 antibody (1:1000), MMP-9 antibody (1:1000), MMP-2 antibody (1:1000), occludin antibody (1:1000), claudin 5 antibody (1:1000), collagen IV antibody (1:2000), ZO-1 antibody (1:4000), β-actin antibody (1:5000), and p-ERK1/2, p-JNK, p-p38, and p-NF-κB p65 antibody (1:1000). Bands were visualized with enhanced chemiluminescence (Proteintech) and photographed using a membrane imaging system (Bio-Rad, Hercules, CA, USA). Band intensity was semi-quantitatively measured by ImageJ software (NIH, Bethesda, MD, USA).
6
Protein Immunoblotting with Antibody Detection
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After sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE), proteins were transferred onto a PVDF membrane. The PVDF membrane was blocked for 2 hours and then incubated with corresponding primary antibodies (Table 1 ) at 4°C overnight. And they were incubated with secondary antibodies ( HRP‐conjugated AffiniPure Goat Anti‐Rabbit IgG (H + L), 1:1000/2000/3000; Proteintech, SA00001‐2; HRP‐conjugated AffiniPure Goat Anti‐Mouse IgG (H + L), 1:6000/1:8000; Proteintech, SA00001‐1) for 2 hours in room temperature in the next day. After washing with PBS for five times, the membranes were detected by enhanced chemiluminescence (Proteintech).
7
Exosome Protein Characterization in AC16 Cells
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The human CM line AC16 was obtained from the cell bank of the Chinese Academy of Science (Shanghai, China). The bicinchoninic acid assay was used to determine the total protein quantity from AC16 cells and exosomes. The proteins were separated on sodium dodecyl sulphate gel and transferred onto the polyvinylidene fluoride membranes. The proteins on the membranes were blocked and probed with primary antibodies against rabbit anti‐CD9 (Proteintech), rabbit anti‐CD81 (Proteintech) and rabbit anti‐Calnexin (Proteintech) overnight at 4°C, followed by incubation with the secondary antibodies against HRP‐lablled sheep anti‐rabbit immunoglobulin (Ig) G (Proteintech) for 1 h at room temperature. Enhanced chemiluminescence (Proteintech) was used to visualize the bands.
8
Western Blot Protein Analysis Protocol
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First, cells were gathered and lysed using radioimmunoprecipitation assay buffer (Beyotime, Jiangsu, China) based on the manufacturer’ protocol. Then the membrane was sealed by 5% skim milk powder for 1 h. Following that, the membrane was incubated with the primary antibodies overnight at 4°C. Then, Tris-buffered saline containing 0.1% Tween-20 (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) was used to wash the membrane three times for 5 min each. The membrane was probed with the secondary antibody for 1 h at room temperature and washed and added enhanced chemi-luminescence (ProteinTech Group, Inc., Chicago, IL, USA). GAPDH was used as the internal control. The QUANTITY ONE software (version 4.6.9; Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to scan the gray value.
9
Western Blot Analysis of FDX1 Protein
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Whole-cell lysates were extracted using RIPA buffer (Applygen Technologies Inc., Beijing, China) supplemented with a protease inhibitor cocktail (GlpBio Technology, Shanghai, China). The protein concentration was measured using a BCA protein assay kit (Elabscience Biotechnology Co., Ltd., Wuhan, China). Cell lysates were performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the protein bands were electrophoretically transferred onto PVDF membranes. Subsequently, non-specific antigen binding was blocked using 5% skim milk. The membranes were incubated at 4°C overnight with primary antibodies (1:1,000-diluted FDX1; Proteintech Group, Inc., Wuhan, China). Membranes were incubated the next day with 1:5,000-diluted secondary antibodies at room temperature for 1 h (Proteintech Group). Enhanced chemiluminescence was used to visualize the image (Proteintech Group). Antibody against β-actin was used as an internal reference (Proteintech Group).
10
Western Blotting and Co-IP Analysis
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Knockdown e ciencies and biochemical responses were analyzed by western blotting. Cells were lysed in RIPA lysis buffer (EMD Millipore Corp.), supplemented with protease inhibitor and phosphatase inhibitor cocktail tablets (Roche). Separated proteins were transferred to nitrocellulose lter membranes and blocked in 5% milk in Tris-buffered saline, with 0.05% Tween-20. Immunodetection was done with various primary antibodies. Appropriate horseradish peroxidase-conjugated secondary antibodies were used and signals were visualized with enhanced chemiluminescence (Proteintech) by Tanon-5200 Chemiluminescent Imaging System (Tanon, China). Cells for co-IP were lysed in lysis buffer (50 mM Tris, pH 7.5, with 150 mM NaCl, 0.5% NP-40, and protease inhibitor and phosphatase inhibitor cocktail tablets (Roche) at 4°C for 30 min. After sonication and centrifugation, cell lysates were incubated with beads (Sigma) at 4°C overnight on a rotator. After six washes with wash buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 0.05% Tween-20, 0.1 mM EDTA), 50 µL of elution buffer (100 mM glycine-HCl, pH 2.5) was added to resuspend the beads, and the eluted proteins were obtained by centrifugation, followed by SDS-PAGE and immunoblotting analysis.
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