5; 20 mM NaCl; 5 mM MgCl2; 1 mM CaCl2; 10 mM NaHCO3; 0.02 % (w/v) β-DDM). The main peaks were pooled and concentrated by ultrafiltration (Vivaspin

20, 100 kDa cutoff) to a volume of 200 μl and when necessary re-injected for a second separation. Absorption spectroscopy and chlorophyll determination Thylakoid protein content was measured referring to the Chl a and Chl b concentrations. The analysis was done photometrically in 80 % (v/v) acetone using a Pharmacia Biotech Ultrospec 4000 spectrophotometer and Chl concentrations were calculated according to Porra et al. (1989). Absorption spectra were recorded EPZ015938 ic50 at room temperature in the range of 370–750 nm with an optical path length of 1 cm and a band-pass of 2 nm. Polyacrylamide gel electrophoresis and western blots For denaturing SDS PAGE, 10 % (w/v) separating polyacrylamide/urea gels with 4 % (w/v) stacking gels were used (Schägger and Jagow 1987). Samples were denatured with Rotiload (Roth)

at room temperature before loading, and after the electrophoretic separation the gels were stained with Coomassie brilliant blue G250. Blue native gel electrophoresis was carried out using 3–12 % (w/v) continuous gradient selleck products gels according to Schägger and Jagow 1991. PSII complexes at 0.2 mg Chl/ml were mixed with 0.25 volumes of Coomassie Blue Solution (5 % (v/v) serva Blue G, 750 mM aminocaproic acid, 35 % Resminostat (w/v) sucrose). Electrophoresis was carried out at 205 V for 5 h at 4 °C. For 2D separation,

the strips from the BN-PAGE were excised and denaturated with Rotiload (Roth) at room temperature for 20 min. After denaturation the strips were placed on the top of a denaturing SDS-PAGE as described above and sealed with Agarose 0.5 % in cathode buffer. For Western blots, gels were first equilibrated in cathode buffer (25 mM Tris/HCl, pH 9.4; 40 mM glycine; 10 % (v/v) methanol). For transfer of the proteins onto a PVDF membrane, filter papers soaked in two different anode buffers (0.3 M Tris/HCl, pH 10.4; 10 % (v/v) methanol and 25 mM Tris/HCl, pH 10.4; 10 % (v/v) methanol) and in cathode buffer were used. Transfer was carried out for 30–60 min, at a current of 1.5 mA/cm2. The membranes were treated with the antisera (purchased from Agrisera, Sweden) solutions, the resulting bands visualized by ECL (Amersham) and signals were recorded on X-ray film (Kodak). Stripping of the antibodies in order to probe one blot with different antibodies was carried out as recommended by the manufacturer of the ECL kit. Mass spectroscopy The in-gel digested samples were analyzed by ESI LC–MS/MS using an HCT ultra ETD II iontrap instrument (Bruker) linked to an Easy nano LC system (Proxeon). selleck kinase inhibitor Processing, deconvolution, and compound detection for the LC–MS/MS datasets were performed using the Data Analysis software (4.0 SP4, Bruker).