Protocols > Protein extraction from grasses

Chloroplast fraction, C4 type plant, 2 types of chloroplasts can be conducted according to the following method: Mechanical Isolation of Bundle Sheath Cell Strands and Thylakoids from Leaves of C4 Grasses. Romanowska and Parys, 2011.

The above mentioned protocol is universal and can be applied to different grasses, such as Echinochloa, Panicum and Zea mays.

Depending on the species, age of the material, growth conditions or variety (for maize), the time of the material shredding must be modified before the BS fraction is isolated. Leaves of Echinochloa and Panicum are more delicate compared to maize, and the time of shredding must thus be shorter. The degree of shredding is assessed using an optical microscope.

Total protein extraction is performed using standard procedures by freezing the material in liquid nitrogen (note: superoxide dismutases, SOD, may lose activity using this type of extraction), and protein extraction in a standard buffer like TIRS or PBS pH 7.5-7.8, containing protease inhibitors, followed by centrifugation to remove debris. Estimation of protein concentration is done using the Bradford method, or commercial reagents that allow estimation of protein concentration in presence of denaturing agents and detergents.

This type of extraction is not most efficient for content of cells in proximity of bundle sheath in C4 plants. Shredding of these cells is limited and the extract will mainly consist of the content of mesophyll cells.

Courtesy of Dr. Wiola Wasilewska-Dębowska, Warsaw University, Poland