RIPA lysis buffer containing 1% PMSF was added into myocardial tissue and the supernatant was harvested by centrifugation (4°C, 12000 rpm, 10 min). Protein concentration was determined using a BCA Protein Assay Kit (Beyotime Institute of Biotechnology, Haimen, China). Protein lysates (40 μg) were separated on SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA, USA). Subsequently, the membranes were blocked with 5% non-fat milk/1% BSA and then incubated overnight at 4°C with cyclooxygenase (COX)-2 antibody (1:1000), IκB antibody (1:1000), NF-κB antibody (1:1000, Wanleibio, Shenyang, China) and p-IκB antibody (1:500, BIOSS, Beijing, China). Then, the membranes were incubated for 45 min at 37°C with HRP-conjugated anti-rabbit IgG antibody (Beyotime Institute of Biotechnology). Protein bands were detected using ECL agent (Wanleibio) and the band intensities were quantified using Gel-Pro-Analyzer Software (Media Cybernetics, Bethesda, MD, USA).
Hrp conjugated anti rabbit igg antibody
Manufactured by Beyotime
The HRP-conjugated anti-rabbit IgG antibody is a laboratory reagent that binds to rabbit immunoglobulin G (IgG) antibodies. The antibody is conjugated with the enzyme horseradish peroxidase (HRP), which enables the detection and visualization of target rabbit IgG in various immunoassay applications.
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Lab products found in correlation
Bca protein assay kit, by Beyotime (2 mentions)
Gel pro analyzer software, by Media Cybernetics (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ecl western blotting detection reagent, by Beyotime (1 mentions)
Phospho mtor, by Cell Signaling Technology (1 mentions)
β actin, by Beyotime (1 mentions)
Gel imaging system, by Bio-Rad (1 mentions)
Quantity one, by Bio-Rad (1 mentions)
2 protocols using hrp conjugated anti rabbit igg antibody
1
Western Blot Analysis of Myocardial Proteins
2
Protein Expression Analysis in Hepatic Samples
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The proteins were isolated from hepatic samples, and their concentrations were measured using BCA Protein Assay Kit (Beyotime Institute of Biotechnology, China). The lysate was mixed with 5× SDS sample buffer and boiled for 10 min. Lysate samples were separated on 6 % and 12 % SDS-polyacrylamide gels, and transferred to a PVDF membrane. The blots were blocked with 5 % milk blocking solution for 2 h at room temperature and then incubated overnight with antibodies against PER1 (1:1,000; Abcam, USA), mTOR (mammalian rapamycin), Phospho-mTOR (1:1,000; Cell Signaling Technology, USA), and β-actin (1:1,000; Beyotime Institute of Biotechnology). HRP-conjugated anti-rabbit IgG antibody (1:1,000; Beyotime Institute of Biotechnology) was used as the secondary antibody. The blots were visualized by ECL Western Blotting Detection Reagents (Beyotime Institute of Biotechnology) and the images were performed by GEL imaging system (Bio-Rad, USA). The quantification of proteins was analyzed by the software Quantity One (Bio-Rad, USA).
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