Isolation of de-enveloped chloroplasts

Spinach (S. oleracea) was purchased from the local grocer. Chloroplasts were isolated in the dark according to an adapted protocol from Caffarri et al. (2009). A handful of leaves was chilled in ice water and then quickly homogenized in a blender in ice cold buffer 1 (B1: 400 mM sorbitol, 5 mM EDTA, 10 mM NaHCO₃, 5 mM MgCl₂, and 20 mM tricine). The resulting suspension was filtered through cheesecloth, and the chloroplasts were pelleted by centrifugation (1,500 × g, 5 min, 4 °C). The pellet was carefully resuspended in buffer 1. Centrifugation and resuspension were repeated twice. This procedure yielded both enveloped and de-enveloped chloroplasts. These buffers were chosen to prevent an osmotic shock for the chloroplasts and potential thylakoid swelling/shrinking. Chloroplasts were kept at 4 °C in the dark until further use. The data of 5 independent chloroplast isolations were used in this study.