Amino acid composition of the purified 20 kDa protein.

The pure 20 kDa protein obtained after concentration with an Amicon cell and using PM10 membrane was then purified by reversed-phase HPLC a using gradient liquid chromatography and a 4.6×75 mm Ultrapore RPSC C8 column. A linear gradient elution was performed in 60 min at a flow rate of 1 ml/min using solvent A: 0.08% trifluoroacetic acid in water, and solvent B: 0.08% trifluoroacetic acid in 60% acetonitrile. Peptide detections were monitored at 220 mm. Such purified 20 kDa preparation was either used as antigen to be injected in rabbit to obtain specific polyclonal antibody directed against the 20 kDa protein, or for amino acid composition determination. Amino acid analyses were performed on protein samples hydrolysed at 110°C under vacuum in 6 M HCl for 24 h using a Beckman Model 332B autoanalyzer. ND: not determined.