5 mg of PSII chlorophylls, i.e., a yield of about 1.4 %. On

the contrary, with the milder protocol B starting from the Dinaciclib nmr same amount of thylakoids only 20 mg of chlorophylls went in solution, i.e., only about 60 % of Chl was recovered. However, from those 20 mg the final amount of PSII chlorophylls harvested was typically 0.4 mg, implying an yield of 2 % of solubilized selleck chemical material or 1.1 % of total Chl. This value is comparable with the recovery observed in protocol A and indicates that the PSII monomeric form is present in roughly the expected amounts judging from total chlorophylls. Subunit composition of the two PSII preparations The two PSII purified batches were next investigated for their subunit composition by denaturing gel electrophoresis and mass spectrometry. The main PSII core subunits were present in both preparations. However, the samples obtained with protocol B contained the PsbS subunit that was totally absent or only present in trace amounts in samples from protocol A, as shown in Fig. 3. Fig. 3 Denaturing SDS-PAGE analysis of PSII preparations according to protocol A (PSII-A) and protocol B (PSII-B). Lane M shows the molecular marker. The labels for protein bands represent the identifications as found by

ESI LC–MS/MS peptide mass finger printing (see Table 1) Further investigation by mass spectrometry (Table 1) shows that protocol A retained four Epacadostat in vivo CAB proteins (CAB2, CAB25, CAB26, CAB36). Both preparations contained significant amounts of the

subunit CP29 (product of the gene Lhcb4), but none of the major LHCII (polypeptides Lchb1-3). Western Blotting using commercially available polyclonal antibodies confirmed the correct assignment of the different subunits (Table 1). These experiments show that the PsbS protein is present in much higher abundance in B than A samples and that the major LHCII are missing in both preparations. Based on these findings, we will refer to the dimeric fraction obtained from protocol A as PSIId, the monomeric fraction as PSIIm and the monomeric fraction, enriched in PsbS obtained from protocol B as PSIImM (where M stand for Mild). Western blots on the BN-PAGE and on its second dimension SDS-PAGE were performed in order to check whether the presence of PsbS in the PSIImM samples was Chloroambucil actually due to the binding, or if it was just the result of a co-migration with PSII monomers. In both cases an anti-PsbS reaction was only observed at the level of PSII monomers, neither in dimers nor as a single PsbS protein. However, when performing BN-PAGE followed by western blotting on thylakoids obtained by protocol B, diffuse signals starting from masses of 360 kDa until 20 kDa were obvious (data not shown). Moreover, we observed also that the single-band obtained from the BN-PAGE on PSIImM samples appeared composite when resolved in second dimension SDS-PAGE (Fig. 2c).