Peroxidase conjugated goat anti rabbit igg secondary antibody

Leaf samples for soluble protein extraction were harvested from the youngest fully expanded leaf at the mid-tillering stage between 09:00 h and 11:00 h, and stored on ice immediately. Leaves were homogenized to a fine powder using a nitrogen-cooled mortar and pestle. Proteins were extracted and fractionated by SDS-PAGE as described previously (Lin et al., 2016 (link)). Samples were loaded based on equal leaf area (0.2364 mm² for ZmPEPC and ZmPPDK, and 2.364 mm² for ZmNADP-MDH, ZmNADP-ME and OsGDCH). After electrophoresis, proteins were electroblotted onto a polyvinylidene difluoride membrane and probed with rabbit antisera against ZmPEPC, ZmNADP-MDH, ZmNADP-ME (all provided by Richard Leegood, University of Sheffield, United Kingdom), ZmPPDK (provided by Chris Chastain, Minnesota State University, United States), and OsGDCH protein (provided by Asaph Cousins, Washington State University, United States). The dilutions of ZmPEPC, ZmPPDK, ZmNADP-MDH, ZmNADP-ME, and OsGDCH antisera were 1:20,000, 1:20,000, 1:5,000, 1:2,000, and 1:100, respectively. A peroxidase-conjugated goat anti-rabbit IgG secondary antibody (Sigma-Aldrich, United States)² was used at a dilution of 1:5,000 and immunoreactive bands were visualized with ECL Western Blotting Detection Reagents (GE Healthcare, United Kingdom)³.