FLAG-affinity pulldown experiments with sll1214 and sll1874 from Synechocystis

Putative subunits of the Mg-protoporphyrin monomethylester oxidative cyclase from Synechocystis PCC 6803, sll1214 and sll1874 were expressed with N-terminal 3xFLAG-tags in separate transformants.. Solubilised thylakoid membranes from these 2 transformants were incubated with anti-FLAG agarose and after extensive washing, the FLAG-tagged proteins, together with putative interaction partners were desorbed with FLAG peptide. The eluates were denatured, reduced, alkylated and digested with trypsin. The resulting peptides were cleaned by cation exchange and desalted, then analysed by nanoLC-MS/MS. The FLAG pulldown experiment was also carried out with a thylakoid membrane preparation from wild-type with no FLAG-tagged proteins as a control. Protein identification was by database searching with Mascot.