Cultured cells were extracted and lysed for 60 min at 4 °C in cell lysis buffer (Intron Biotechnology, Seoul, Republic of Korea). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on equal amounts of proteins (30–40 μg) using 10% gel. The extracted proteins were separated and put to a nitrocellulose membrane (Amersham Bioscience, Westborough, MA, USA). The transferred membranes were probed with the corresponding primary antibodies, followed by binding with secondary antibodies linked to horseradish peroxidase (Santa Cruz Biotechnology). An enhanced chemiluminescence kit was used to detect immunoreactive signals (Amersham Bioscience).
Secondary antibodies linked to horseradish peroxidase
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Secondary antibodies linked to horseradish peroxidase are reagents used in various immunological techniques, such as Western blotting and enzyme-linked immunosorbent assay (ELISA). They serve as detection tools, binding to the primary antibody and enabling visualization or quantification of the target analyte through the enzymatic activity of horseradish peroxidase.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Cytiva (1 mentions)
Enhanced chemiluminescence kit, by Cytiva (1 mentions)
Cell lysis buffer, by iNtRON Biotechnology (1 mentions)
Nephrin, by Abcam (1 mentions)
Image acquisition and analysis software, by Bio-Rad (1 mentions)
Podocin, by Abcam (1 mentions)
Sirt1, by Abcam (1 mentions)
Cleaved caspase 3, by Abcam (1 mentions)
Hif 1α, by Novus Biologicals (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
2 protocols using secondary antibodies linked to horseradish peroxidase
1
Western Blot Analysis of Proteins
2
Renal Protein Expression Analysis
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Samples were randomly selected from the six groups. Assays were performed thrice. Kidney cortex tissue specimens underwent homogenization, followed by a 10-minute centrifugation (16000×g at 4°C). A bicinchoninic acid protein assay kit (Pierce Co, Rockford, IL, USA) was utilized for protein quantitation. Totally 20 µg of protein per sample underwent separation by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and transfer onto polyvinylidene difluoride (PVDF) membranes. After blocking (5% skimmed milk in Tris-buffered saline), overnight incubation was carried out with primary antibodies targeting collagen I (1:1000; Abcam, Cambridge, MA, USA), cleaved caspase-3 (1:1000; Abcam), podocin (1:1000; Abcam), nephrin (1:1000; Abcam), HIF-1α (1:1000; Novus Bio, Littleton, CO, USA), SIRT1 (1:1000; Abcam) and β-actin (1:1000; Cell Signaling, Danvers, MA, USA). Next, a 2-hour incubation with secondary antibodies linked to horseradish peroxidase (1:5000; Santa Cruz, CA, USA) was carried out. Quantitation was performed by densitometry with the image acquisition and analysis software (Bio-Rad).
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