IC4R010-Proteomic-2004-14730683

Project Title

• Proteome analysis of cultivar-specific deregulations of Oryza sativa indica and O. sativa japonica cellular suspensions undergoing Rice yellow mottle virus infection

Protein Extraction and 2-D PAGE

• Aliquots (1 g) of synchronized cell samples were ground to a fine powder in liquid nitrogen. Protein extraction and precipitation was performed in 10% v/v TCA in acetone with 0.07% v/v b-mercaptoethanol at 2207C for 1 h, followed by centrifugation for 10 min at 47C, 50 0006g. The pellets were washed twice with cold acetone containing 0.07% b-mercaptoethanol and centrifuged for 10 min at 47C, 50 0006g, and the pellets dried. Proteins were solubilized in 500 mL of lysis buffer (9 M urea, 4% w/v CHAPS,0.5% v/v Triton X-100, 0.5% IPG buffer, 3 mM tributylphosphine (TBP)). Finally, the sample was centrifuged 10 min at 207C, 60 0006g. Concentration of the supernatant was determined using protein assay (2D-Quant kit; Amersham, UK).