/We prepared the total membrane of spinach leaves first and then used a two-phase separation method to remove the majority of heavily stacked thylakoid membranes. The bottom fraction of the two-phase method contains dominant amount of photosynthesis related complexes, including Photosystem I, Photosystem II, ATP synthase, Cytochrome b6f complex, etc. The top membrane fractions are collected, pelleted, resuspended, and washed thoroughly with a sucrose-rich buffer. This step could efficiently remove most of the soluble protein/complexes (house-keeping enzyme complexes), nucleic acid containing complexes (ribosomes, chromosomes, etc.) and less well-structured proteins for example cell skeletons. Purified membrane fractions were pelleted using ultracentrifugation, resuspended, aliquoted, flash frozen in liquid nitrogen and stored at −80 °C until further use.