Protocol: Phenol/Ammonium Acetate-Methanol Precipitation
This method can be very useful for solubilizing proteins from plant membranes.
- Suspend membrane pellet in 0.5 ml of extraction buffer (0.7 M sucrose, 0.5 M Tris, 30 mM HCl, 50 mM EDTA, 0.1 M KCl , 2% (v/v) 2-mercaptoethanol and 2 mM PMSF).
- Homogenize with a glass homogenizer fitted with a Teflon plunger.
- Adjust sample to 1.5 ml with extraction buffer. Incubate for 10 min at 4°C.
- Add an equal volume of water-saturated phenol. Shake at room temperature for 10 min.
- Separate the phases by centrifugation.
- Recover the phenol phase and re-extract with an equal volume of extraction buffer.
- Precipitate proteins from the phenol phase by addition of 5 volumes of 0.1 M ammonium acetate in methanol. Incubate at -20°C overnight.
- Wash the precipitate 3 times with ammonium acetate in methanol and 1 time with acetone. Air dry the pellet.
- Solubilize in rehydration buffer by incubation for 1 hour at room temperature.
- Remove insoluble material by centrifugation (178,000 x g for 15 min).
Reference: Westermeier, Reiner (2006) Preparation of plant samples for 2-D electrophoresis, Practical Proteomics 1-2, 56-60.