Cell lysates were made by resuspending cells in three cell pellet volumes of RIPA buffer (50 mM Tris-Cl, pH 7.4,150 mM NaCl, 2 mM EDTA, 0.5% sodium deoxycholate, 1% Nonidet P-40 and 0.1% SDS) containing protease inhibitors (Complete Mini Protease Inhibitor Cocktail; Roche, Indianapolis, IN, USA). Lysates were resolved by SDS-PAGE. The proteins were transferred to PVDF (Bio-Rad, Hercules, CA, USA) and immunodetected using appropriate primary and peroxidase-coupled secondary antibodies (GE Healthcare Life Sciences, Pittsburg, PA, USA). Proteins were visualized by West Pico and West Dura Chemiluminescence Substrate (Pierce, Rockford, IL, USA).
Primary and peroxidase coupled secondary antibodies
Manufactured by GE Healthcare
Sourced in United States
Primary and peroxidase-coupled secondary antibodies are laboratory reagents used in various immunological techniques. Primary antibodies are specific to a target antigen, while the peroxidase-coupled secondary antibodies bind to the primary antibodies, enabling detection and visualization of the target. These antibodies are commonly used in techniques such as Western blotting, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry to identify and quantify target proteins.
Automatically generated - may contain errors
2 protocols using primary and peroxidase coupled secondary antibodies
1
Western Blot Protein Quantification
2
Protein Expression and Detection Protocol
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Cells were treated as indicated. Cells were lysed in IP-lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.1% SDS, 1% NP-40 containing protease inhibitors (cOmplete Mini Protease Inhibitor Cocktail). Protein concentration was determined by BCA assay (Thermo Fisher, Waltham, MA, USA). 25-35 µg total protein (lysate) or 30-60 µg culture media (supernatant) was resolved by SDS-PAGE and transferred onto 0.45 µm nitrocellulose membrane (Thermo Fisher, Waltham, MA, USA), and immunodetected using appropriate primary and peroxidase-coupled secondary antibodies (GE Healthcare Life Sciences, Pittsburg, PA, USA). Proteins were visualized by West Pico and West Dura chemiluminescence Substrate (Thermo Fisher, Waltham, MA, USA).
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