For total protein extraction and western blot analysis, cells were lysed in ice-cold RIPA buffer [50 mM Tris-Cl (pH 8.0), 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl] supplemented with protease inhibitors (Roche). The lysate was incubated on ice for 30 min and sonicated with ice-cooling for 3 × 5 s pulses at a frequency of 5 microns using a Soniprep 150 plus (MSEs) followed by centrifugation at 13,000 × g for 10 min at 4°C. The amount of protein was quantified using the Pierce BCA Protein Assay Kit (ThermoFisher). Typically, 50 μg of total protein in Laemmli buffer [2% SDS, 10% glycerol, 50 mM Tris-HCl (pH 6.8), bromophenol blue 0.02%, 1% β-mercaptoethanol] was heated to 95°C for 5 min and separated by SDS-PAGE and subsequently transferred to Amersham Protran 0.2 μm nitrocellulose membrane (GE Healthcare). The membrane was blocked with 5% non-fat dry milk in TBST [20 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.1% Tween 20] and probed with indicated primary antibodies overnight at 4°C. Membranes were then washed with TBST three times for 10 min at room temperature followed by incubation for 1 h with the appropriate secondary antibody. The membrane was washed again three times and signals were detected using ECL Western Blot Substrate (BioRad) and the luminescent image analyser LAS-4000 (Fujifilm).
Ecl western blot substrate
Manufactured by Bio-Rad
Sourced in United States
The ECL Western Blot Substrate is a chemiluminescent detection reagent used in Western blot analysis to visualize and quantify proteins. It utilizes a luminol-based system to produce a light signal proportional to the amount of target protein present on the membrane.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Bca assay, by Beyotime (2 mentions)
Amersham protran 0.2 μm nitrocellulose membrane, by GE Healthcare (1 mentions)
Las 4000, by Fujifilm (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
β actin, by ABclonal (1 mentions)
Gs 800, by Bio-Rad (1 mentions)
6 protocols using ecl western blot substrate
1
Total Protein Extraction and Western Blot Analysis
2
Quantitative Western Blot Analysis of MEF Lysates
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RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 % NP−40, 0.5% sodium deoxycholate, and 0.1 % SDS) containing 1x cOmplete protease inhibitor cocktail (Roche) was used to collect total cellular lysates from MEFs. Pierce BCA protein assay kit (ThermoFisher Scientific) was used to measure protein concentration in all samples. 20 μg of total protein per sample together with 1x Laemmli buffer was loaded to a 10% SDS-polyacrylamide gel electrophoresis (PAGE) gel and separated under reducing conditions. The separated proteins were transferred to a polyvinylidene difluoride membrane, after which the membrane was blocked with 3% milk in 1x TBST buffer (20 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween 20) for 1 hr. Immunoblotting with primary antibodies was performed overnight at 4 °C in a rotator, followed by 3 washes with 1x TBST, subsequent incubation with HRP-fused secondary antibodies for 4 h at 4 °C in a rotator, and 3 washes with 1xTBST. Protein bands were developed with ECL Western Blot Substrate (BioRad) and western blots were imaged by a ChemiDoc MP Imaging system (BioRad). The quantification of protein bands was performed by ImageJ software by comparing the signal from each antibody to the GAPDH signal which served as a loading control. Four blots were used for the quantification of each sample.
3
Protein Extraction and Western Blot
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Total proteins of tissue samples and cells were extracted by RIPA lysis buffer with 1mM PMSF (Beyotime, China). After quantified by BCA assay (Beyotime, China), proteins were separated using 10% SDS-PAGE gel and transferred to PVDF membrane. Membranes were blocked in 5% BSA in TBST for 1h at room temperature, then incubated with primary antibodies overnight at 4 °C. Subsequently, Membranes were incubated with HRP-conjugated secondary antibodies for 1h at room temperature, and developed using ECL Western Blot Substrate (Bio-rad, USA). Details of primary antibodies were listed in Supplementary file 1: Table S3 .
4
Quantification of Synaptic Proteins in Prefrontal Cortex
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Tissues of the prefrontal cortex were homogenized in ice-cold RIPA lysis buffer, supplemented with EDTA, protease inhibitor cocktail, and phosphatase inhibitor. The homogenate was sonicated six times for 4 seconds, at 6 seconds intervals on ice, and then centrifuged at 3864 g for 20 min at 4°C. The supernatant was collected, and the protein concentration was quantitated by BCA assay (Beyotime, P0010). Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. The membrane was blocked with 5% non-fat milk (2.5 g skim milk powder + 50 ml washing buffer) at room temperature for 1 h and then incubated with the primary antibody at 4°C overnight. These primary antibodies included: rabbit anti-synaptophysin (Abcam, ab32127), rabbit anti-PSD95 (Invitrogen, 51–6900), and β-actin (ABclonal, AC026). Following 3 washes in TBST, the membrane was incubated with HRP-inked anti-rabbit IgG secondary antibody (CST, 7074) at room temperature for 1 h. After washing 3 times with TBST, the protein bands were detected with ECL western blot substrate (Bio-Rad, 1, 705,060) and visualized using the ChemiDoc Touch imaging system (Bio-Rad).
5
Western Blot Analysis of MEF Lysates
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Total lysate of MEFs were collected in RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) with protease inhibitors, using the Pierce BCA (12-Bicinchonic acid) protein assay kit (Thermo Fisher Scientific) was performed on samples and 20 µg of protein per sample was loaded to the 10% SDS-polyacrylamide gel electrophoresis (PAGE) gel. The extracts were separated under reducing conditions and transferred to polyvinylidene difluoride membrane. After staining with primary antibodies and subsequent washes in 1× TBST buffer (20 mM Tris, 150 mM NaCl, 0.1% Tween-20), immunoblots were stained with HRP-fused secondary antibodies. Protein bands were developed with ECL Western Blot Substrate (Bio-Rad) and imaged by a ChemiDoc MP Imaging system (Bio-Rad). The quantification of the blots was performed by ImageJ software and at least three blots were used for analysis for each samples.
6
Western Blot Analysis of Lupeol and NF-kB
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Treated (Lupeol and Lupeol+IR) HEp-2 cells were homogenized and lysed in western blot lysis buffer (15mM Tris, 2mM EDTA, 50mM 2-Mercaptoethanol, 20% Glycerol, 0.1% Triton X100, 1mM PMSF, 1mM Sodium Fluoride, 1mM Sodium Orthovanadate, 1µg/ml Aprotinin, 1µg/ml Leupeptin, 1µg/ml Pepstatin) at room temperature. Fifty microgram of total cell lysates (TLC) were separated on a 10% sodium dodecyl sulphate-polyacrylamide gel SDS-PAGE, and blotted onto nitrocellulose membranes, blocked in TBS-T (0.1 % Triton in 1xTBS) and probed with anti-AKT, NF-kB and COX-2 primary antibody overnight at 4ºC. The membranes were then incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies. The immunoreactive protein bands were developed by enhanced chemiluminescence kit (BioVision ECL Western Blot Substrate, USA) and analyzed by a densitometer (Bio Rad, GS 800, USA) for quanti cation.
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