A simple and efficient method to prepare pure dimers and monomers of the cytochrome b6f complex from spinach
Using a single size-exclusion chromatography we were able to isolate highly pure dimers and monomers of the Cyt b6f complex from spinach from a bulk preparation of that protein complex obtained with a standard procedure. At higher protein/detergent ratio during the chromatography most of the Cyt b6f complex remained as dimers. In contrast, at lower protein/detergent ratio (around 15 times lower), most dimers became monomerized. As a bonus, this chromatography also allowed the elimination of potential Chl a contaminant to the Cyt b6f preparations. SDS-PAGE protein analysis with 18% (w/v) acrylamide revealed the loss of the ISP subunit in our monomeric preparation. However, it fully retained the content of Chl a, a prerequisite to perform any spectroscopic study involving this unique pigment.
KeywordsCytochrome b6f Photosynthesis Plant Protein Purification SDS-PAGE
This work was supported by the Ministry of Economy and Competiveness of Spain (Grant No. AGL2014-55300-R) and Aragon Government (Grant E33).
- Bald D, Kruip J, Boekema EJ, Rögner M (1992) Research in photosynthesis. In: Murata N (ed) Proceedings of the IXth international congress on photosynthesis. Kluwer Academic Publishers, Nagoya, pp 629–632Google Scholar
- Vener AV, van Kan PJ, Rich PR, Ohad I, Andersson B (1997) Plastoquinol at the quinol oxidation site of reduced cytochrome bf mediates signal transduction between light and protein phosphorylation: thylakoid protein kinase deactivation by a single-turnover flash. Proc Natl Acad Sci USA 94:1585–1590CrossRefPubMedPubMedCentralGoogle Scholar