The total proteins were extracted from tissues and cultured cells in ice-old RIPA lysis buffer (Beyotime Institute of Biotechnology), and the protein concentrations were determined using a BCA protein assay kit (Beyotime Institute of Biotechnology). Total proteins (30 µg/lane) of each experimental group were subjected to 10% SDS-PAGE and then transferred onto polyvinylidene fluoride membranes (EMD Millipore). After blocking in TBS containing 5% skimmed milk for 1 h at room temperature, the membranes were incubated at 4°C overnight with primary antibodies against VOPP1 (1:1,000; Sigma-Aldrich; Merck KGaA; cat. no. HPA038371) and GAPDH (1:1,000; Sigma-Aldrich; Merck KGaA; cat. no. SAB1410512). Subsequently, the membranes were incubated with the secondary antibody conjugated with peroxidase (1:2,000; Sigma-Aldrich; Merck KGaA; cat. no. AP510) for 1 h at 37°C. Protein bands were detected using the enhanced chemiluminescence detection system (Bio-Rad Laboratories, Inc.) and Quantity One software v4.6.2 (Bio-Rad Laboratories, Inc.).
Secondary antibody conjugated with peroxidase
Manufactured by Merck Group
Secondary antibody conjugated with peroxidase is a laboratory reagent used in various immunoassay techniques. It serves as a detection agent, binding to a primary antibody and enabling the visualization or quantification of target analytes through an enzymatic reaction.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Beyotime (2 mentions)
Quantity one software v4, by Bio-Rad (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Enhanced chemiluminescence detection system, by Bio-Rad (1 mentions)
Mmp13, by Cell Signaling Technology (1 mentions)
Col2a1, by Merck Group (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
2 protocols using secondary antibody conjugated with peroxidase
1
Protein Extraction and Western Blot Analysis
2
Quantification of Extracellular Matrix Proteins
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The total proteins in cells were extracted and the protein concentrations were determined by BCA protein assay kit (Beyotime). 30 μg of the total proteins were applied to 10% SDS-PAGE and then transferred onto PVDF membranes. After being blocked in TBS containing 5% non-fat milk for 1 h, the membranes were co-incubated with the primary antibodies at 4°C overnight against MMP-13 (1:1,000 dilutions, Cell signaling technology, Cat. no.69926), Col2a1 (1:1,000 dilutions, Sigma, Cat. no.SAB4500366), and GAPDH (1:1,000 dilutions, Cell signaling technology, Cat. no.5174), and then with the secondary antibody conjugated with peroxidase (1:2000 dilutions, Sigma-Aldrich, Cat. no. AP510). Protein bands were detected using the enhanced chemiluminescence detection system.
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