Differential Proteome and Secretome Analysis During Rice–Pathogen Interaction - Plant Proteomics: Methods and Protocols - page 544

Fig. 2 Workfl ow of secretome analysis during rice-pathogen interaction
6. Add 1 M sucrose, vortex, and centrifuge at 5,000 × g for 15 min
at RT, collect supernatant phenol phase, and transfer new tube.
7. Add 4 volumes of 100 mM ammonium acetate in methanol to
precipitate secreted proteins for overnight at −20 °C.
8. Centrifuge at 5,000× g for 10 min, wash and resuspend
precipitated protein in 80 % methanol containing 0.1 mM
ammonium acetate. The washing step was repeated three times.
9. Wash with 80 % acetone and stored the precipitated secretory
proteins in the same solution at −20 °C.
10. Proceed for 2-DE ( see Note 11 ) or MudPIT analysis
( see Note 12 ).