Lung samples were homogenized in ice-cold RIPA buffer (50 mmol/L Tris, 150 mmol/L NaCl, 1% Triton X-100, pH 7.4) containing protease inhibitor cocktail (Roche Life Science Co., USA). The whole cell lysates were obtained by centrifuging the suspensions for 10 min at 13,000 ×g at 4°C. The lysates were loaded onto a 10% SDS-polyacrylamide gel for separation and then transferred to nitrocellulose membranes. The blots were probed with anti-p-Jun N-terminal kinase (JNK) (1:400; Santa Cruz Biotechnology, Inc., USA), anti-JNK (1:400; Santa Cruz Biotechnology, Inc., USA), and anti-β-actin (1:400; Santa Cruz Biotechnology, Inc., USA) antibodies. After incubation with horseradish peroxidase-linked secondary antibodies, specific protein bands on the blots were visualized with the ECL chemiluminescence detection system (Millipore, USA) according to the manufacturer's manual.
Ecl chemiluminescence detection system
Manufactured by Merck Group
Sourced in United States
The ECL chemiluminescence detection system is a laboratory equipment used to detect and quantify the presence of specific proteins or other molecules in a sample. The system utilizes a chemiluminescent reaction to generate light, which is then detected and measured. This allows for highly sensitive and precise analysis of target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Anti jnk, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Nf κb p65, by Abcam (1 mentions)
Phospho nf κb p65 p p65, by Abcam (1 mentions)
Cd206, by Abcam (1 mentions)
Lamin b1, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Gapdh, by Abcam (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Semi dry system, by Bio-Rad (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
4 protocols using ecl chemiluminescence detection system
1
Western Blot Analysis of Lung Proteins
2
Western Blot Analysis of Cellular Proteins
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Proteins extracted from BV2 cells by RIPA buffer containing the protease and
phosphatase inhibitors (Roche) were used for western blotting. In brief, protein
samples (30 µg/lane) were separated on 12% SDS-PAGE and transferred to PVDF
membrane (Millipore). Membranes were blocked with fat-free milk (5%) in TBST for
2 h at room temperature. Membranes were then respectively incubated with the
primary antibodies overnight at 4°C, including iNOS (1:500, Abcam), CD206
(1:1000, Abcam), TLR4 (1:500, Abcam), NF-κB p65 (1:2000, Abcam), phospho-NF-κB
p65 (p-p65, 1:2000, Abcam), IKBα (1:1000, Abcam), phospho-IKBα (p- IKBα, 1:2000,
Abcam), GAPDH (1:2000, Abcam), LaminB1 (1:2000, Abcam). After washing 3 times
with TBST, membranes were respectively incubated with HRP-conjugated secondary
antibodies (1:4000) for 2 h at room temperature. GAPDH and LaminB1 were used as
the internal control. Membranes were detected by the ECL chemiluminescence
detection system (Millipore). The band intensity was measured using Image J.
phosphatase inhibitors (Roche) were used for western blotting. In brief, protein
samples (30 µg/lane) were separated on 12% SDS-PAGE and transferred to PVDF
membrane (Millipore). Membranes were blocked with fat-free milk (5%) in TBST for
2 h at room temperature. Membranes were then respectively incubated with the
primary antibodies overnight at 4°C, including iNOS (1:500, Abcam), CD206
(1:1000, Abcam), TLR4 (1:500, Abcam), NF-κB p65 (1:2000, Abcam), phospho-NF-κB
p65 (p-p65, 1:2000, Abcam), IKBα (1:1000, Abcam), phospho-IKBα (p- IKBα, 1:2000,
Abcam), GAPDH (1:2000, Abcam), LaminB1 (1:2000, Abcam). After washing 3 times
with TBST, membranes were respectively incubated with HRP-conjugated secondary
antibodies (1:4000) for 2 h at room temperature. GAPDH and LaminB1 were used as
the internal control. Membranes were detected by the ECL chemiluminescence
detection system (Millipore). The band intensity was measured using Image J.
3
Western Blot Analysis of Protein Expressions
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Western blotting was performed essentially as described previously [40 ]. Briefly, cells were harvested and washed with ice-cold PBS and lysed in 1xRIPA buffer containing 1 mM DTT and Complete Mini EDTA-free protease inhibitor cocktail and incubated on ice for 30 minutes. Cell lysates were clarified by centrifugation at 14,000 rpm for 15 min at 4°C and protein concentration was measured using protein assay kit (Bio-Rad). Lysates (20 μg/lane) were electrophoresed in 8% or 12% SDS-PAGE gel electrophoresis and transferred onto PVDF membrane by a semi-dry system (Bio-Rad) and reacted with optimal concentrations of specific primary antibodies at 4°C overnight followed by secondary antibodies conjugated with HRP (Cell Signaling) for 1 hour at room temperature. Visualization of immunoreactive proteins was achieved with ECL chemiluminescence detection system (Millipore, Billerica, MA). Alpha-tubulin was used as loading control. Similarly, xenograft tumor tissues harvested from mice were homogenized in 2% SDS lysis buffer and processed for Western blotting as described above. Immunoblotting data represent contiguous lanes.
4
Hippocampal Adenosine Receptor Quantification
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The hippocampus tissue was separated and homogenized in lysis buffer (0.32 M sucrose, 1 mM EDTA, 10 mM HEPES, 1 mg/ml BSA, 0.1 mM PMSF, pH 7.4). After the homogenate was centrifuged at 3,000 g at 4°C for 10 min, the supernatant was centrifuged at 16,000 g at 4°C for 60 min, and the precipitate was re-suspended in 5% (W/V) SDS. Next, the protein extracts were electrophoretically separated by 10% SDS polyacrylamide gel electrophoresis, and then the gel was transferred to a 0.45 μm nitrocellulose (NC) membrane at 4°C. After blocking non-specific sites with TBST (Tris Buffered Saline with Tween 20) containing 5% defatted dried milk, the membranes were incubated with the antibodies of A1R (1:1,000) and A2AR (1:1,000) overnight at 4°C, and γ-tubulin antibody (1:5,000) blotting was used as a loading control. The membranes were incubated with goat anti-mouse and goat anti-rabbit secondary antibodies in 5% defatted dried milk with TBST at room temperature for 1 h. Analysis was performed using an ECL chemiluminescence detection system (Millipore, USA) and the band intensity was quantified with Image J software (National Institutes of Health, Bethesda, MD, USA).
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