Quantitative analysis of leaf photosynthetic proteins

Soluble protein was extracted from frozen leaf material of 21-d-old plants (sixth and seventh leaf) in protein extraction buffer [20 mM Tris–HCl pH 7.5 with 5 mM MgCl₂, 300 mM NaCl, 5 mM DTT, 1% Triton X-100, and cOmplete Mini EDTA-free Protease Inhibitor Cocktail (Roche)]. Samples were heated at 80 °C for 15 min with 1× Bolt LDS sample buffer (ThermoFisher Scientific) and 200 mM DTT. Extracts were centrifuged and the supernatants subjected to SDS-PAGE on a 12% (w/v) polyacrylamide gel and transferred to a nitrocellulose membrane. Membranes were probed with rabbit serum raised against wheat Rubisco at 1:10,000 dilution (52 (link)), the SSU RbcS2 from Chlamydomonas (CrRbcS2) [raised to the C-terminal region of the SSU (KSARDWQPANKRSV) by Eurogentec] at 1:1,000 dilution, ACTIN (AS21 4615, Agrisera) at 1:1,000 dilution, SAGA1 at 1:1,000 dilution (20 (link)), SAGA2 (raised against the peptide sequence SRNGTHAAGEDVREV by Eurogentec) at 1:1,000, and EPYC1 at 1:2,000 dilution (17 (link)), followed by HRP-linked goat anti-rabbit IgG (Abcam) or HRP-linked rabbit anti-mouse (Agrisera) at 1:10,000 dilution, and visualized using Pierce ECL Western Blotting Substrate (Life Technologies).