Lysates were quantified using the Bradford method (Bio-Rad) and heated at 95°C for 5 min. Lysates were fractionated by SDS-PAGE and transferred to PVDF membranes (Bio-Rad) in 25 mM Tris, 192 mM glycine, 0.01% SDS, and 20% methanol. Membranes were blocked in 1× TBS containing 1% Tween-20 and 5% BSA (blocking solution) for 1 h at room temperature with gentle rocking. Membranes were then incubated overnight at 4°C with TnC (RRID:AB_306435; Abcam), TnI (RRID:AB_2206278; Cell Signaling), TnT (RRID:AB_261723; Sigma-Aldrich), tropomyosin (RRID:AB_261817; Sigma-Aldrich), desmin (RRID:AB_306653; Abcam), actinin (RRID:AB_476766; Sigma-Aldrich), and α-actin (RRID:AB_476695; Sigma-Aldrich) primary antibodies, SERCA2a (Abcam), phospholamban (RRID:AB_2617049; Badrilla), and phosphor-PLB (RRID:AB_310352; Millipore) followed by secondary antibodies after washing. Blots were developed using ECL Plus Western blotting detection reagents (Merck). The relative densities were calculated by normalizing each blot to actin. The number of experiments means each different animal. We excluded the results that showed large differences in internal control (10%) between LV and RV protein samples.
Ecl plus western blotting detection reagents
Manufactured by Merck Group
Sourced in United States
ECL Plus Western blotting detection reagents are a chemiluminescent detection system used for the sensitive and quantitative analysis of proteins in Western blot applications. The reagents produce a luminescent signal proportional to the amount of target protein, allowing for the visualization and quantification of protein expression levels.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (7 mentions)
Bca kit, by Thermo Fisher Scientific (6 mentions)
Ripa lysis buffer, by Beyotime (4 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Nap1l1, by Abcam (2 mentions)
Ccnd1, by Abcam (2 mentions)
C jun, by Proteintech (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Tropomyosin, by Merck Group (1 mentions)
α actin, by Merck Group (1 mentions)
Desmin, by Abcam (1 mentions)
Serca2a, by Abcam (1 mentions)
Bradford method, by Bio-Rad (1 mentions)
Radioimmunoprecipitation assay lysis buffer, by CWBIO (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Nf κb, by Abcam (1 mentions)
Ap124p, by Merck Group (1 mentions)
29 protocols using ecl plus western blotting detection reagents
1
Western Blot Analysis of Cardiac Proteins
2
Western Blot Analysis of Oxidative Stress Markers
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Cell lysates were prepared using radioimmunoprecipitation assay lysis buffer (CW Biotech) in the presence of a protease inhibitor cocktail (Thermo Fisher Scientific, Inc.). Protein concentrations of cell lysates were quantified using a Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Inc.) according to the manufacturers protocol. Total protein (10 ug) were loaded in each well of 12% sodium dodecyl sulfate polyacrylamide gel and subjected to electrophoresis. The proteins were then transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk for 1 h at room temperature and incubated with primary antibodies against GAPDH (1:5,000; cat. no. 10494-1-AP; ProteinTech Group, Inc.), Nrf2 (1:1,000; cat. no. ab62352; Abcam), HO-1 (1:1,000; cat. no. ab13243; Abcam) and NF-κB (1:10,000; cat. no. ab16502; Abcam) at 4°C overnight, followed by incubation with a horseradish peroxidase-conjugated secondary antibody (1:5,000; cat. no. SA00001-2; ProteinTech Group, Inc.) for 1 h at room temperature. Detection was performed using ECL Plus western blotting detection reagents (EMD Millipore) and the blots were semi-quantified using ImageJ 2 (National Institute of Health).
3
P-glycoprotein Expression in HepG2 Cells
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Lysates of HepG2 cells (which were treated with 5‐Aza‐2‐DC and tacrolimus) were run on 8% sodium dodecyl sulfate‐polyacrylamide gels, transferred to polyvinylidene difluoride membranes, and incubated with an antibody against P‐glycoprotein (Abcam Inc. Cambridge, MA, USA). Antibody binding was detected using enhanced chemiluminescence ECL Plus western blotting detection reagents (Merck Millipore Ltd. Tullagreen, Carrigtwohill, Co. Cork, Ireland).
4
Investigating RBM10 Regulation in A549 and H1299 Cells
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A549 and H1299 cells were transfected with pcDNA3.1-RBM10 wild type or mutated cDNA or vector-only for 48 h. Total cellular protein was then extracted using the radioimmunoprecipitation assay lysis buffer and protein concentrations were measured using bicinchoninic acid (BCA) kit (CWBiotech, Beijing, China). Equal amounts of protein samples (20 µg each) were heated at 100°C for 10 min and then separated in 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred onto a polyvinylidenedifluoride (PVDF) membrane (EMD Millipore). The membranes were incubated in 5% bovine serum albumin (BSA) for 2 h at the room temperature and then incubated with primary antibodies at a dilution of 1:1,000 against Numb (1:1,000; cat. no: 18701-1-AP), Notch-1 (1:1,000; cat. no: 10062-2-AP), Fas (1:1,000; cat. no: 13098-1-AP), E-cadherin (1:1,000; cat. no: 20874-1-AP), and CyclinD1 (1:1,000; cat. no: 60186-1-Ig; all from ProteinTech Group, Inc., Chicago, IL, USA) at 4°C overnight. On the next day, the membranes were washed with Tris-based saline-Tween 20 solution (TBST) three times and then incubated with a secondary antibody at a dilution of 1:1,000 (1:1,000; cat. no: AP124P; EMD Millipore) for 1 h at the room temperature. Protein bands were visualized using enhanced chemiluminescence (ECL)-Plus western blotting detection reagents (EMD Millipore).
5
Gestational Immune Profile Analysis
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Total protein was extracted from spleen tissues isolated from five pregnant CBA/J mice from each group on day 14 of gestation using RIPA lysis buffer (Beyotime Institute of Biotechnology). Protein (30 µg/lane) was quantified using a bicinchoninic acid assay kit (Beyotime Institute of Biotechnology) and separated by 10% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 10% skim milk at room temperature for 4 h. Subsequently, the membranes were incubated at 4°C overnight with the following primary antibodies: Anti-FoxP3 (cat. no. bs-0269R; 1:1,000; BIOSS), anti-TNF-α (cat. no. A0277; 1:1,000; ABclonal Biotech Co., Ltd.), anti-IFN-γ (cat. no. bs-0480R; 1:1,000; BIOSS), anti-IL-4 (bs-0581R; 1:1,000; BIOSS) and anti-β-actin (cat. no. AC026; 1:2,000; ABclonal Biotech Co., Ltd.). Subsequently, the membranes were incubated with a goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (1:2,000; cat. no. bs-0295G-HRP; BIOSS). Protein bands were visualized using ECL Plus Western Blotting Detection reagents (EMD Millipore). Blots were performed in triplicate and protein expression was quantified using ImageJ 2× software (National Institutes of Health) with β-actin as the loading control.
6
Western Blot Protein Quantification
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The extracted proteins were separated by 10% SDS-PAGE and further transferred onto PVDF membranes (Millipore, Bedford, MA, United States). Antibodies including NAP1L1 (Abcam), CCND1 (Abcam), HDGF (Proteintech), and c-Jun (Proteintech) were used in the Western blot assays following the manufacturer’s instructions. Detection was performed using ECL Plus Western blotting detection reagents (Millipore, United States). The specific protein expression levels of the blots were normalized to GAPDH (Santa). The Cat numbers, origins, and dilution concentrations used for all antibodies are listed in Supplementary Table 2 .
7
Protein Expression and Western Blotting
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The cells were lysed with cell lysis buffer (Beyotime P0013B) supplemented with the protease inhibitor complex (Beyotime P1006). The nuclear and cytoplasmic proteins were extracted using a nuclear and cytoplasmic protein extraction kit (Beyotime P0027) following the manufacturer’s protocols. The protein concentrations in the extracts were detected using the BCA assay (Beyotime P0012S). Equal amounts of the protein sample were loaded to sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels. Then, the protein sample was electrotransferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% BSA dissolved in TBST (50 mM Tris/HCl, pH 7.6, 150 mM NaCl, and 0.1% Tween-20) for 2 h at room temperature and incubated with the indicated primary antibody overnight at 4 °C, followed by incubation with appropriate enzyme horseradish peroxidase-linked secondary antibody for 2 h at room temperature. Protein bands were visualized using ECL plus western blotting detection reagents (Millipore, MA, USA). The blot images were captured using the FluorChem8000 imaging system (Alpha-Innotech, CA, USA). The gray values were analyzed using ImageJ gel analysis software [21 (link)].
8
Western Blot Analysis of VEGFA
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Total protein was separated by SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE). The proteins in SDS‐PAGE were transferred onto nitrocellulose membranes (GE Healthcare). The membrane was incubated with rabbit anti‐VEGFA (ab46154, 1:1000 dilution; Abcam) primary antibodies overnight at 4°C and then with secondary antibody at room temperature for 1 hour. Proteins of interest were visualized using ECL Plus Western blotting Detection Reagents (Millipore).
9
Quantifying BRCA1 Protein Expression
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Total cell lysates were extracted using RIPA lysis buffer (Beyotime, China) and quantified using the BCA protein assay kit (Beyotime, China). Equal amounts of proteins were separated using 8% SDS-PAGE and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The membranes were blocked in 5% skim milk for one hour and then incubated overnight at 4°C with the following primary antibodies: HA-Tag (C29F4) rabbit mAb (1 : 1000 dilution; Cell Signaling Technology, USA), GAPDH (D16H11) XP rabbit mAb (1 : 1000 dilution; Cell Signaling Technology, USA). HA-Tag (C29F4) rabbit mAb was used for detecting labeled BRCA1 protein. Then, the membranes were incubated again with goat anti-rabbit IgG/goat anti-mouse IgG horseradish peroxidase- (HRP-) conjugated secondary antibodies (1 : 2000 dilution, Cell Signaling Technology, USA) for two hours at room temperature. Protein bands were visualized by using the ECL Plus western blotting detection reagents (Millipore, Billerica, MA, USA).
10
Western Blot Protein Analysis Protocol
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Proteins were extracted, and 50 μg proteins were electrophoresed on SDS polyacrylamide gels with Tris-glycine running buffer and transferred onto 0.45 mm PVDF membranes. After blocking with 5% (w/v) bovine serum albumin (BSA) in Tris-buffered saline (TBS) for 2 h at room temperature, the membrane was washed four times using TBST (TBS and 0.5% Tween-20) and then incubated with primary antibodies overnight at 4°C. After washing, the membrane was incubated with HRP-anti rabbit IgG for 2 h. Protein bands were visualized by ECL Plus western blotting detection reagents (Millipore, USA). GAPDH was used as an internal control. Each blot was a representative of three independent experiments, and band intensity was measured using ImageJ software.
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