Abstract
This rapid protocol allows the extraction of chloroplast enriched proteins from Nicotiana benthamiana (N. benthamiana) leaves that were transiently transformed to express an epitope tagged protein of interest. Thus, it can serve as a tool to study the chloroplastidic localization of the protein of interest when it is combined with western-blot analysis. Agrobacterium-mediated transformation (Agroinfiltration, Romeis et al., 2001) is used to transiently express a protein carrying an epitope tag in tobacco leaves. Here, co-infiltration with an Agrobacterium strain harboring 19 K from soil-borne wheat mosaic virus suppresses posttranscriptional gene silencing and therefore increases transformation efficiency (Te et al., 2005).The chloroplast isolation of the transformed leaves is based with modifications on Romeis et al. (2001), and includes mechanical breakage of cell wall and membranes, the removal of unbroken tissue by filtration and the separation of intact chloroplasts by centrifugation through a Percoll layer.
Materials and Reagents
Equipment
Procedure
Representative data
Figure 1. Agroinfiltration procedure. A. The leaves indicated with number 3, 4 and 5 are best used for agroinfiltration. B. The infiltrated areas are marked with a marker pen. Figure 2. Separation of intact from broken chloroplasts after centrifugation through a 40% Percoll layerl Figure 3. A Intact chloroplasts and B broken chloroplast visualized by phase contrast microscopy
Recipes
Acknowledgments
This protocol was adapted from previous work (Romeis et al., 2001; Te et al., 2005; Pineda et al., 2010). I would like to acknowledge R. Deeken (Department of Molecular Plant Physiology and Biophysics, University of Wuerzburg, Germany) and the DFG Research Training Group 1342 (University of Wuerzburg, Germany) for funding. In addition, I would like to than C.W. Lee for help and advice.
References
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