Protocols > Phenol protein extraction

Procedure

1. 1 g of fresh leaf tissue was grinded in a mortar using liquid nitrogen to form a fine powder.
2. The powder was resuspended in 3 ml of high density extraction buffer (0.5 M Tris-HCl, 0.7 M sucrose, 1 mM PMSF, 50 mM EDTA, 0.1 M KCl and 0.2% β-mercaptoethanol; pH 8.0).
3. The homogenate was transferred to a conic tube and was mixed in an orbital shaker on the ice during 10 min. 1 ml of saturated phenol on Tris-HCl (pH 6.6-8.0) were added and the homogenate was mixed in an orbital shaker on the ice during 10 min.
4. The mixture was centrifuged at 4 ºC during 10 min at 5000 xg, the organic phase was recovered on a new tube and 4 volumes of ammonium acetate 0.1 M on methanol were added, the solution was mixed in a vortex and was stored at -20ºC overnight.
5. The mixture was centrifuged at 4500xg for 15 min at 4ºC and pellet was washed twice with ammonium acetate 0.1 M on methanol and one with acetone, the pellet was dried at room temperature.
6. Proteins were resuspended to saturation in Tris-HCl (50 mM, pH 8.0) and quantified according Bradford methodology (Bradford, 1976).