from the transgenic rice plants were used to isolate vrd1 protein more specifically one hundred grams of dry seeds harvested from the primary transgenic rice lines were ground in a mill the seed powder was extracted in 500 ml of an extraction buffer 10 mm nah 2 po 4 15 mm na 2 hpo 4 500 mm nacl 100 mm kcl 2 mm edta 1 5 polyvinylpolypyrrolidine and 1 mm phenylmethylsulfluoride ph 6 4 at 4 u00b0 c for 16 hours under continuous stirring solid materials were subsequently removed by centrifugation at 11 000 u00d7 g for 30 minutes solid ammonium sulfate was then added to the supernatant to obtain 30 80 saturated ammonium sulfate fraction 30 80 as fraction the 30 80 as fraction was dissolved in distilled water and dialyzed against 20 mm tris hcl ph 7 0 buffer a the dialyzed solution was passed through a cm sepharose cl 6b pharmacia column 2 6 cm u00d7 30 cm previously equilibrated with buffer a the column was washed with 10 volume of buffer a and eluted with a linear gradient 800 ml total volume of 0 0 5 m nacl in buffer a fractions 12 ml each were collected at a flow rate of 4 ml minute proteins in the fractions were monitored at 280 nm aliquots of protein fractions were analyzed on nupage 4 12 bis tris gel invitrogen fractions having a protein band of about 5 1 kda were pooled and concentrated with a masterflex ultrafiltration apparatus millipore the concentrated solution was further purified by fplc on a superdex peptide hr 10 30 column pharmacia and eluted with buffer a containing 150.