Proteins were resolved by SDS-PAGE, and gels were transferred to polyvinylidene difluoride membrane (GE Healthcare). Membranes were washed with TBS 0.2% Tween, blocked in TBS supplemented with 0.2% Tween, 3% BSA and 0.5% gelatin for 30 min at room temperature and probed with specific antibodies overnight at 4°C. After three washes of 10 min in TBS 0.2% Tween, blots were incubated with horseradish peroxidase-linked secondary antibody (Jackson ImmunoResearch) for 45 min at room temperature, followed by EZ-ECL assay, according to the manufacturer's instructions (Biological Industries). Chemiluminescence was detected using a Chemidoc (BioRad). Primary antibodies used were: anti-Srebp2 (ab30682; Abcam), p-AMPKα (#2535; cell signaling), Abca1 (ab18180; Abcam), Abcg8 (ab126493; Abcam) and anti-Gapdh (ab9485; Abcam).
Horseradish peroxidase linked secondary antibody
Manufactured by Jackson ImmunoResearch
Sourced in United States, Panama, China
Horseradish peroxidase-linked secondary antibodies are a type of lab equipment used in immunoassays. They function as signal-amplifying conjugates, binding to primary antibodies and reacting with a substrate to produce a measurable signal.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (5 mentions)
Bca assay, by Cell Signaling Technology (4 mentions)
Polyvinylidene difluoride membrane, by Merck Group (3 mentions)
Enhanced chemiluminescence system, by GE Healthcare (3 mentions)
Ripa buffer, by Cell Signaling Technology (3 mentions)
Anti mus81, by Santa Cruz Biotechnology (3 mentions)
Ecl plu, by Cytiva (3 mentions)
Anti gapdh, by Merck Group (3 mentions)
G box, by Syngene (2 mentions)
Beclin 1, by Cell Signaling Technology (2 mentions)
Anti rad52, by Santa Cruz Biotechnology (2 mentions)
Anti gfp, by Santa Cruz Biotechnology (2 mentions)
Lamin b1, by Abcam (2 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (2 mentions)
Image lab 6, by Bio-Rad (2 mentions)
Mab3291, by R&D Systems (2 mentions)
Chemidoc imaging system, by Bio-Rad (2 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (2 mentions)
Anti smarcal1, by Fortis Life Sciences (2 mentions)
Anti zranb3, by Proteintech (2 mentions)
27 protocols using horseradish peroxidase linked secondary antibody
1
Western Blot Analysis of Lipid Regulators
2
Adipose Tissue AKT Phosphorylation
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Protein extracts from peritoneal macrophages and splenocytes, and western blotting were performed as previously described with antibodies against Id3 (Calbioreagents) and β-tubulin (Cell Signaling Technology). 10 (mg) omental adipose tissue was homogenized in 250ul protein lysis buffer (10% glycerol, 1% NP-40, 137 mM NaCl, 25 mM HEPES pH 7.4, 1 mM EGTA) containing protease inhibitors and phosphatase inhibitors (Sigma–Aldrich) and lysed on ice for 30 min. Protein lysate was collected and western blotting was performed with antibodies against AKT (1:1000, Cell Signaling) and Thr308 pAKT (1:1000, Cell Signaling), followed by horseradish peroxidase-linked secondary antibody (Jackson). Relative AKT phosphorylation was determined by normalizing pAKT to total AKT in each sample.
3
Protein Expression Analysis in Osteoblastic Cells
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Total protein was extracted from OS cell lines and human normal osteoblastic cell line hFOB 1.19 using the RIPA buffer containing protease inhibitor cocktail (Sigma, USA), and 90 μg of total protein per well was used for Western blot in accordance with the protocols of the manufacturer. The primary antibodies of c-Fos (1:1000, Cell Signaling), Wnt2 (1:1000, Abcam), and Fzd9 (1:1000, Abcam) incubated at 4°C for overnight and the appropriate horseradish peroxidase-linked secondary antibody (1:20000, Jackson Immuno-Research, USA) were used to visualize the immunoreactivity. The house-keeping protein GAPDH was used as an internal control. The intensity of each band was measured with the Image-Pro Plus 6.0 analytic system.
4
Western Blot Protein Detection Protocol
5
Apoptosis Signaling Pathway Profiling
6
Western Blot Analysis of Apoptosis Markers
7
Autophagy Pathway Protein Analysis
8
Western Blot Analysis of Testes Proteins
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Testes were lysed with SDS sample buffer and heated for 5 min at 95°C. Samples were separated by SDS–polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA, USA), followed by blocking in tris-buffered saline with Tween 20 (TBST) containing 5% defatted milk (BD Biosciences, Franklin Lakes, NJ, USA) for 30 min. After probing with primary antibodies, the membranes were washed in TBST, incubated with a horseradish peroxidase–linked secondary antibody (Jackson ImmunoResearch Laboratories) for 1 hour, followed by three washes with TBST. Bound antibodies were detected using the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific Inc., Waltham, MA, USA). The primary antibodies and dilution factors used are listed in table S3.
9
Protein Extraction and Western Blotting
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Whole proteins from cell lysates and lung tissues were extracted using RIPA lysis solution (P0013B, Beyotime, Shanghai, China) according to the manufacturer’s instructions and measured using a BCA kit (P0009, Beyotime, Shanghai, China). Equal amounts of protein were separated on 8%–12% SDS–PAGE gels (Vazyme, Nanjing, China), blotted onto PVDF membranes (Merck Millipore, Germany), blocked with 5% nonfat milk in Tris-buffered saline with Tween-20 (TBST) (Beyotime, Shanghai, China) for 1 h, and incubated overnight at 4°C in antibody diluent (Beyotime, Shanghai, China) containing the following primary antibodies: rabbit antibody against p21 (1:1,000 dilution), rabbit antibody against p16 (1:1,000 dilution), rabbit antibody against p-p38MAPK (Thr180/Tyr182) (1:1,000 dilution), rabbit antibody against p38MAPK polyclonal antibody (1:1,000 dilution); mouse antibody against p53 (1:5,000 dilution); mouse antibody against GAPDH (60004-1-Ig, 1:50,000 dilution; ProteinTech); and mouse antibody against ß-Tubulin (10094-1-AP, 1:2,000 dilution; ProteinTech). The membranes were incubated with horseradish peroxidase-linked secondary antibody (Jackson Labs, United States) for 1 h, and protein expression was detected using ECL (Vazyme, Nanjing, China). Images were captured using the Tanon 5,200 imaging system (Shanghai, China). ImageJ software (version 2.0.0) was used for gray value analysis.
10
Western Blotting for Protein Analysis
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Western blot analyses were described in a previous study [23 (link)]. Briefly, cells were harvested by low-speed centrifugation and washed with PBS. Cells were lysed in RIPA Buffer (Cell Signaling Technology, 9800), and protein concentrations were determined using BCA assays (Cell Signaling Technology, 7780). Forty micrograms of each protein sample were separated using 12% SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad). Membranes were blocked with 1 × Tris-buffered saline with Tween 20 (TBST) and 5% (w/v) non-fat milk for 1 h at room temperature. Then, membranes were incubated with primary antibodies overnight at 4 °C. Subsequently, blots were incubated with horseradish peroxidase-linked secondary antibodies (Jackson ImmunoResearch) for an additional 1 h. Immunoreactive bands were visualized using an enhanced chemiluminescence system (GE Healthcare).
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