Equal volumes of denatured BALF from the different experimental groups were loaded onto 4–20% Criterion TGX polyacrylamide gels (Bio-Rad, Hercules, CA) for electrophoresis under reducing conditions. Proteins were transferred onto PVDF membranes (Bio-Rad, Hercules, CA), and probed with antibodies against YM1 (STEMCELL Technologies) and FIZZ1 (Abcam, Cambridge, MA). Secondary antibodies conjugated to HRP were used to detect target proteins, which were visualized with the ECL method (Bio-Rad). Chemiluminescence from the blots was captured using KwikQuant Imager (Kindle Bioscience, LLC) and target band intensities were measured using the Image Studio Lite software (LI-COR Biosciences, NE).
Ecl method
Manufactured by Bio-Rad
Sourced in United States
The ECL (Enhanced Chemiluminescence) method is a laboratory technique used for the detection and quantification of target proteins in Western blot analysis. It relies on the emission of light through a chemiluminescent reaction to visualize the presence and abundance of specific proteins in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Image studio lite software, by LI COR (1 mentions)
Criterion tgx polyacrylamide gel, by Bio-Rad (1 mentions)
Fizz1, by Abcam (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Polyvinylidene difluoride pvdf membrane, by Merck Group (1 mentions)
Hrp labeled goat anti rabbit igg, by Beyotime (1 mentions)
Anti h3k9me2 antibody, by Abcam (1 mentions)
Anti h3 antibody, by Abcam (1 mentions)
Horseradish peroxidase hrp labeled goat anti mouse igg, by Beyotime (1 mentions)
Pvdf membrane, by Roche (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Mouse monoclonal antibody against β actin, by Merck Group (1 mentions)
Anti rabbit igg hrp linked antibody, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Bca assay, by Beyotime (1 mentions)
Bca protein assay kit, by Bio-Rad (1 mentions)
Peroxidase conjugate secondary antibodies, by Santa Cruz Biotechnology (1 mentions)
8 protocols using ecl method
1
Quantifying Lung Protein Expression
2
Western Blot Protein Analysis Protocol
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According to previously reported methods (4 (link),12 (link)), the total protein samples of all groups were subjected to 12% SDS-PAGE denaturing gel electrophoresis (Bio-Rad) and transferred onto polyvinylidene difluoride PVDF membranes (Millipore, Bedford, MA, USA). After the membranes were blocked and washed, primary antibodies were added and incubated at 37°C for 45 min (Table II ). Then, after the membranes were fully washed, secondary antibodies were added and incubated at 37°C for 45 min (Table II ). The membranes were washed with Tris-buffered saline containing Tween-20 (TBST, Bio-Rad) at room temperature four times for 14 min per wash. The results were developed using the enhanced chemiluminescence (ECL) method (Bio-Rad). The membranes were exposed using Kodak XAR-5 films (Sigma-Aldrich).
3
Whole-cell Protein Extraction and Immunoblotting
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Whole-cell protein extracts were prepared from HeLa cells lysed in extraction buffer (100 mM Tris–HCl pH 7.4, 50 mM NaCl, 5 mM EDTA, and 1 mM PMSF). Proteins were loaded onto a 12% SDS-PAGE gel and separated by electrophoresis. Then, proteins were transferred to PVDF membranes (Millipore, Billerica, MA, United States) and the membranes were incubated with 5% non-fat milk-TBS for 2 h at room temperature. Afterward, the immunoblots were incubated overnight with the following primary antibodies: anti-H3 antibodies (Abcam, Cambridge, United Kingdom, ab1791), anti-H3K9me2 antibodies (Abcam, Cambridge, United Kingdom, ab1220), anti-GAPDH antibodies (Beyotime, Shanghai, China), and anti-RNase H1 monoclonal antibodies (Abcam, Cambridge, MA, United States, ab56560). The secondary antibodies were horseradish peroxidase (HRP) labeled goat anti-mouse IgG (Beyotime, Shanghai, China, A0126) and HRP labeled goat anti-rabbit IgG (Beyotime, Shanghai, China, A3327). Immunoreactivity was determined using the ECL method (Bio-Rad, Hercules, CA, United States) according to the manufacturer’s instructions.
4
Western Blot Protein Detection Protocol
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It was separated for 2 hours on a 10% SDS-PAGE gel before being transferred to a PVDF membrane (0.45 μm, Roche). The membrane was blocked for 1 hour at 37 °C with 5% TBST, then the primary antibody was added and incubated for 8 hours at 4 °C. The membrane was then rinsed three times with TBST before being incubated for 1 h at 37 °C with HRP-conjugated goat anti-rabbit or goat anti-mouse immunoglobulin G secondary antibody (Jackson ImmunoResearch Laboratories). TBST washed the membrane three times more. The target western blot was detected using the ECL method (Bio-Rad, USA). Proteintech provided the CDK2(22060–1-AP) and p21(10355–1-AP) antibodies used in Western blotting in this investigation, while Cell Signaling Technology provided the Rb(#9313) and p-Rb(#8516) antibodies.
5
Protein Expression Analysis in Breast Cell Lines
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Analysis of the protein expression was performed as described previously [55 (link)]. Briefly, total cell lysates from breast cell lines were prepared in lysis buffer (20% SDS, glycerol, 1 M Tris (pH 6.8)) containing protease inhibitor cocktail (Sigma-Aldrich, USA). An equal amount of proteins (10 µg) were loaded and separated in 8% SDS-PAGE or 3–8% Tris–acetate gel (for CBP only) and then transferred into nitrocellulose membrane (Sigma-Aldrich, USA). Followed by immunoblotting with rabbit monoclonal primary antibodies against CBP (#7389), GCN5 (#3305), ERα (#13258) and HER2 (#4290) (Cell Signaling Technology, USA) at dilution 1:1000 and mouse monoclonal antibody against β-actin (#A5441) at dilution 1:2000 (Sigma-Aldrich, USA), then with secondary anti-rabbit IgG, HRP-linked antibody and anti-mouse IgG, HRP-linked antibody (Cell Signaling Technology, USA). The detection of membrane was carried out by the ECL method (Biorad, USA) and developed using ChemiDoc™ imaging system (Biorad, USA). The band quantification was carried out using Image Lab™ software (Biorad, California, USA) with β-actin as loading control.
6
IGF2 Protein Expression Analysis by Western Blot
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The protein expression of IGF2 was determined by western blotting. Briefly, total protein was extracted using RIPA lysis buffer and protein concentration was determined by BCA assay (Beyotime Institute of Biotechnology). Proteins (30 µg/lane) were separated by SDS-PAGE on 12% gel and transferred onto a PVDF membrane (Millipore). Following blocking with 5% skimmed milk for 1 h at room temperature (~25°C), the PVDF membrane was incubated with primary antibodies against IGF2 (1:1,000, cat. no. ab9574; Abcam) and GAPDH (1:1,000, cat. no. ab8245; Abcam)_at 4°C overnight. Following washing with Tris-buffered saline containing 0.05% Tween-20, horseradish peroxidase-conjugated secondary antibody IgG (H&L, 1:4,000, cat. no. ab6728; Abcam) was added and incubated at room temperature (~25°C) for 1 h. Protein bands were visualized using the enhanced chemiluminescence ECL method (Bio-Rad Laboratories, Inc.) and analyzed with ImageJ software v.1.6.0 (National Institutes of Health).
7
Tissue Protein Extraction and Western Blot Analysis
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Briefly, ultrasonic lysis of skin tissue in RIPA lysate with PMSF, samples were further homogenized in a lysis buffer and centrifuged at 10,000 g, 4 °C for 15 min to collect the supernatant. Protein concentrations were determined by a BCA protein assay kit (Bio-Rad). Subsequently, the supernatant was diluted in 5× SDS loading buffer and boiled for 15 min. The protein samples were separated with 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride filter (PVDF) membranes (Millipore), which were blocked in 5% milk dissolved in Tris buffered saline containing 0.05% Tween 20. Thereafter, the membranes were incubated with primary antibodies at first and then the secondary antibody linked with horseradish peroxidase for 2 h. The detection of chemiluminescent signals was performed by ECL method (Bio-Rad). The Antibodies are listed in Supplementary Table S2 .
8
Western Blotting of Phospho-β-catenin
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A total of 30 μg equivalent amount of protein per sample were loaded on 10% SDS–PAGE gel and transferred to nitrocellulose membranes for further processing. The membrane was blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature, followed by overnight incubation at 4 °C with the primary antibodies [phospho β-catenin Ser552 (CST #9566) (1:500), β-catenin (Abcam #ab32572) (1:500)]. Membranes were incubated with appropriate peroxidase-conjugate secondary antibodies (Santa Cruz, USA, 1:3000) and bands were visualized by the enhanced chemiluminescence (ECL) method (BioRad, USA).
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