Polyclonal antibodies

To determine the content of proteins: ascorbate peroxidase (APX), catalase (CAT), rubisco large subunit (RbcL) and photosystem’s proteins, like: PsbA, Lhcb1 and PsbO plant tissue (shoots) were extracted in buffer according to Laureau et al. [84 (link)] with modifications, in 4 °C. Protein concentration was determined according to Bradford [85 (link)] with BSA as a standard. SDS-PAGE were performed at 4 °C, with 24 mA for 15 min, 34 mA for 35 min and 68 mA for 60 min, on 12% polyacrylamide gels. For each line 5 µg of protein extract was applied. Blotting was performed on a polyvinylidene fluoride membrane (PVPD) (BioRad). Identification of protein bands was done with polyclonal antibodies (Agrisera), and detection was performed with alkaline phosphate buffer with BCIP/NBT as a substrate. Membranes were scanned, and the intensity of bands was calculated using ImageJ version 1.52n (open source software, OSS). The content of each protein was expressed as arbitral units, defined as area under the curve per µg of protein applied to the line. After analysis, the highest protein quantities at each gel were expressed as 1, and all data were normalized to it.

Protein samples were solubilized in SDS sample buffer, separated on SDS-PAGE and electro transferred onto polyvinylidene fluoride membranes. Antibodies were added, and the protein-antibody complexes were labeled using a chemiluminescence reagent (Luminata Crescendo Western HRP Substrate; Merck kGaA). The signals were detected with ChemiDoc analyzer (Bio-Rad Laboratories). The antiserum against FZL was produced by immunizing rabbits with a purified recombinant FZL (60-475 aa) protein (PhytoAB INC). Specific antibodies against CURT1A and RbcL were kindly provided by Chanhong Kim (CAS Center for Excellence in Molecular Plant Science) and Hiroshi Shimada (Hiroshima University), respectively. The antibodies against Tic110, D1 and VIPP1 were described previously (Zhang et al., 2012 (link); Kato and Sakamoto, 2014 (link)) For detection of photosynthetic proteins PsaA, PetA, CF₁β, CF₁γ, PsbS, Lhcb1 and Lhca1, commercially available polyclonal antibodies were used (Agrisera).