RIPA lysis buffer (Beyotime, Beijing, China) was used to extract total protein from cells, and a BCA protein assay kit (Beyotime) was used to measure the protein concentrations following the manufacturer’s protocols. The protein samples were separated and transferred using 12% SDS-PAGE and PVDF membranes. The membranes were blocked in skimmed milk (5%) at room temperature for 2 h. After that, the membranes were cultured with primary antibodies at 4 °C overnight. Primary antibodies includes anti-Smad3 antibody (1:1000, Abcam), anti-ALP antibody (1:1000, Abcam), anti-Osteopontin (OPN) antibody (1:1000, Abcam), anti-Runx-2 antibody (1:1000, Abcam), anti-Osterix antibody (1:1000, Abcam), and anti-GAPDH antibodies (1:2000, Abcam). In the next day, the membranes were incubated with a horseradish peroxidase-conjugate secondary antibody (1:2000, Santa Cruz) for 1 h at room temperature. The enhanced chemiluminescence detection system (ECL, Roche Molecular Biochemicals) was used to measure the blots.
Horseradish peroxidase conjugate secondary antibody
Manufactured by Santa Cruz Biotechnology
Horseradish peroxidase-conjugate secondary antibody is a laboratory reagent used in immunoassays and Western blotting techniques. It consists of a secondary antibody that has been coupled with the enzyme horseradish peroxidase. This enzyme can catalyze a colorimetric or chemiluminescent reaction, allowing for the detection and visualization of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Anti smad3 antibody, by Abcam (1 mentions)
Anti osterix antibody, by Abcam (1 mentions)
Anti alp antibody, by Abcam (1 mentions)
Anti runx2 antibody, by Abcam (1 mentions)
Anti gapdh antibody, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
α sma, by Wanlei (1 mentions)
Chemiscope 3000, by Clinx (1 mentions)
2 protocols using horseradish peroxidase conjugate secondary antibody
1
Protein Expression Analysis in Cells
2
Evaluating p10/OA Complex Effects on Cell Markers
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HSC-T6 cells were plated in a six-well plate at a density of 5.0 × 10 5 cells per well and cultured overnight in an incubator. For 24 h, cells were treated with various molar ratios (8:16 and 8:20) of preformed p10/OA complex. After three times washing with cold PBS, cells were lysed using RIPA lysis buffer containing the protease inhibitor phenylmethylsulfonyl fluoride (PMSF). Equal quantities of protein (approximately 10-20 μg) were separated by 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% nonfat milk in TBST for 1 h, the PVDF membrane was incubated overnight at 4 °C with rabbit polyclonal α-SMA (Wanleibio; 1:1000) and mouse monoclonal COL1α2 primary antibody (Servicebio; 1:1000). Following TBST washes 3 times, the PVDF membrane was incubated for 1 h at room temperature with horseradish peroxidase conjugate secondary antibody (Santa Cruz biotechnology; 1:3000). The Clinx ChemiScope 3000 mini enhanced chemiluminescence (ECL) detection reagent was used to detect a chemical reaction light signal.
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