Protocol for chloroplast purification and import assay in Arabidopsis

Chloroplasts are fundamental plant organelles in green plants and algae. As an endosymbiosis-originated organelle, most chloroplast proteins are encoded by nuclear genes, translated in the cytosol, and imported into the chloroplast. Here, we present a protocol for the purification of Arabidopsis chloroplasts and in vitro translation of the desired protein. We also describe steps for import system preparation, import reaction, and Western blot. This approach eliminates centrifugation, thermolysis, and radioisotope labeling steps and reduces sample preparation time and cost by approximately 50%. For complete details on the use and execution of this protocol, please refer to Zhao et al.¹.