Isolation of cell wall proteins from Medicago sativa stems

Plant cell walls are highly dynamic and chemically active components of plant cells. Cell walls consist primarily of polysaccharides, with proteins comprising approx 10% of the cell wall mass. These proteins are difficult to isolate with a high degree of purity from the complex carbohydrate matrix. This matrix traps proteins and is a source of contamination for subsequent 2-DE analysis. Mature plant tissues provide a further challenge owing to the formation of secondary walls that contain phenolic compounds. This chapter discusses protein extraction from cell walls and presents a specific method for the isolation of proteins from Medicago sativa stem cell walls. The method includes cell disruption by grinding, copious washes with both aqueous and organic solutions to remove cytosolic proteins and small molecule contaminants, and two different salt extractions that provide a highly enriched cell wall protein fraction from alfalfa stem cell walls. Following treatment with a commercial clean-up kit, the protein extracts yield high-quality and high-resolution 2-DE separations from which proteins can be readily identified by mass spectrometry.