Goat anti rabbit igg horseradish peroxidase hrp

Total protein from 2- and 3-DAP seeds of A619 and vks1 was extracted with radioimmunoprecipitation assay lysis buffer (Leagene, catalog number PS0013). Proteins were quantified by Bradford assays. Twenty micrograms of protein was separated by a 10% SDS-PAGE gel and then transferred electrophoretically to a PVDF membrane (Bio-Rad, catalog number 162-0177). The membrane was incubated with VKS1 antibodies at 4°C overnight and then incubated with the secondary antibody, goat anti-rabbit IgG-horseradish peroxidase (HRP; Abmart, catalog number M21002L). To detect the control protein ACTIN, a primary antibody, mouse monoclonal ACTIN antibody (Abmart, catalog number M20009L), and a secondary antibody, goat anti-mouse IgG-HRP (Abmart, catalog number M21001L), were used to perform the immune reaction. The dilution of antibodies was 1:1000. Finally, the membrane was treated with HRP chemiluminescent substrate reagent (Invitrogen, catalog number WP20005) and imaged using a Tanon-5200 system (Tanon).
To analyze the protein content of VKS1 in vks1-2, protein was extracted from the 2-DAP seeds of B73 and vks1-2.

The nonzein protein extraction was according to the standard method of our laboratory^{42 (link)}. Twenty μg of nonzein proteins were used for immunoblotting analysis following the manufacture’s protocol, (Bio-Rad, catalog number 162-0177). VEN1 and the control protein ACTIN antibodies were diluted 1:1000, while the secondary antibodies, goat anti-rabbit IgG-horseradish peroxidase (HRP) (Abmart, catalog number M21002L) and goat anti-mouse IgG-HRP (Abmart, catalog number M21001L), were diluted 1:5000 for performing the immune reaction. The membrane was treated with HRP chemiluminescent substrate reagent (Invitrogen, catalog number WP20005) and imaged using a Tanon-5200 system (Tanon).

The non-zein protein was extracted from 15 DAP kernels of WT and the rcl1 mutant kernels as described previously (Zheng et al., 2019 (link)). An amount of 15 μg of non-zein proteins were separated by 12% (w/v) SDS-PAGE gel and transferred to a Hydrophobic PVDF Transfer Membrane (Millipore, Burlington, MA, USA). Immunoblot analysis was performed as previously described. All of the primary antibodies of the starch synthesis pathway were related (Zhang et al., 2016 (link)). The mouse monoclonal anti-FLAG antibody (Sigma, St. Louis, MO, USA, Cat# F1804) and ACTIN antibody (Abclonal, Woburn, MA, Cat# AC009) were purchased. The secondary antibodies were goat anti-rabbit IgG-horseradish peroxidase (HRP) or goat anti-mouse IgG-HRP (Abmart, Berkeley Heights, NJ, USA, Cat# M21001L) and were diluted 1 : 5000 for performing the immune reaction. The signals were detected using the High-sig ECL Western Blotting Substrate (Tanon, Shanghai, China, Cat# 180-506) and were visualized using the Tanon-5200 imaging system (Tanon, Shanghai, China)