Ultrapure buffer saturated phenol

Proteins were extracted using a previously described phenol-based procedure [44 (link)]. Leaves were ground in a mortar and pestle in liquid nitrogen with 1 % (w/w) PVPP. One hundred mg plant material was resuspended in 600 μL extraction buffer (0.7 M Sucrose, 0.1 M KCl, 0.5 M Tris–HCl pH7.5, 0.5 M EDTA, 1 mM PMSF and 2 % β-mercaptoethanol). Samples were homogenized twice (one min each) with a MM300 TissueLyser (Qiagen). An equal volume of UltraPure Buffer-Saturated Phenol (Invitrogen) was added and the mixture was rehomogenized as described above. After centrifugation at 12,000 × g for 15 min at 4 °C, the upper phenol phase was eliminated and the pellet used for re-extraction in the same buffer. Protein was precipitated from the phenol phase using five volumes saturated ammonium acetate (100 mM) in methanol overnight at −20 °C followed by centrifugation at 12,000 × g for 15 min at 4 °C. Pellets were washed four times with four mL saturated ammonium acetate (100 mM) in methanol and dried 10 min. Proteins were dissolved in urea buffer (7 M urea, 2 M thiourea, 40 mM Tris, 2 % Chaps and 18 mM DTT). The protein concentration was determined using Bradford’s method with BSA as a standard.