Western blot analysis was performed as described previously [28 (link)]. In brief, protein cell lysates (30 μg), were subjected to SDS polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane (BioRad, Watford, UK), and immunoblotted with the following antibodies: anti-CYP27A1 (1:1000, ab227248; Abcam, Cambridge, UK), anti-liver-X-receptor-β (LXR-β) (1;1000, D6M9D; Cell Signalling Technology, Danvers, MA, USA), fibronectin (1:500, 611,447; BD Biosciences, Franklin Lakes, NJ, USA), E-cadherin (1:1000, 610,181; Cell Signalling), vimentin (1:500, 550513; BD Biosciences), ER-β (1:1000, PPZ0506; Thermo Fisher, Waltham, MA, USA), GAPDH (1:5000, CB1001; Millipore, Burlington, MA, USA), β-actin (1:10,000, A1978; Sigma-Aldrich, St. Louis, MO, USA), and ER-α (1:1000, sc-8002; Santa Cruz, Santa Cruz, CA, USA). After incubation with secondary antibodies conjugated to peroxidase (Sigma, St. Louis, MO, USA), proteins were detected with a Clarity ECL substrate (BioRad, Watford, UK) using BioRad Chemidoc XRS + system and quantified using Image software.
Secondary antibodies conjugated to peroxidase
Manufactured by Merck Group
Sourced in United States
Secondary antibodies conjugated to peroxidase are laboratory reagents used in various immunoassay techniques. They function as detection agents, binding to and amplifying the signal from primary antibodies that have recognized their target antigens. The peroxidase enzyme conjugated to the secondary antibody can catalyze a colorimetric or chemiluminescent reaction, allowing for the visualization and quantification of the target analyte.
Automatically generated - may contain errors
Lab products found in correlation
Anti cyp27a1, by Abcam (1 mentions)
Gapdh, by Merck Group (1 mentions)
Clarity ecl substrate, by Bio-Rad (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Fibronectin, by BD (1 mentions)
Vimentin, by BD (1 mentions)
Oxyblot protein oxidation detection kit, by Merck Group (1 mentions)
Caspase 3, by Santa Cruz Biotechnology (1 mentions)
Caspase 9, by Santa Cruz Biotechnology (1 mentions)
2 protocols using secondary antibodies conjugated to peroxidase
1
Western Blot Analysis of Protein Expression
2
Oxidative Stress Protein Profiling
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Total or mitochondrial protein extracts were analyzed by western blot. Polyvinylidene fluoride (PVDF) membranes were incubated with the primary antibodies directed against: cytochrome C, 4-hydroxynonenal (4 HNE), IkBa, caspase-9, caspase-3 (Santa Cruz Biotechnology, Dallas, TX, USA); antimyosin heavy chain (MHC) fast, anti-a tubulin (Millipore, Billerica, MA, USA); nitrotyrosine (Mitosciences); oxidative phosphorylation complexes proteins (OXPHOS; Amersham, England, UK); MnSOD, CuZn SOD (assay designs, Farmingdale, NY, USA); glutathione reductase (GR), and catalase (AbFrontier, Seoul, Korea). After primary antibody incubation, PVDF membranes were incubated with secondary antibodies conjugated to peroxidase (Sigma-Aldrich, St. Louis, MO, USA) and protein expression levels were quantified with ImageJ software corrected with tubulin expression and normalized with CTL set at 100 %. Oxidized proteins were analyzed by western blot with the Oxyblot protein oxidation detection kit (Chemicon International, Temecula, CA, USA).
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