Affinity Purification of Plant Protein Complexes

Proteins were extracted from 4 grams of ∼2.5 week-old above-ground plant tissues ground to a fine powder in liquid nitrogen. The resulting powder was suspended in 14 ml lysis buffer (50 mM Tris HCl pH 7.6, 150 mM NaCl, 5mM MgCl₂, 10% glycerol, 0.5 mM Dithiothreitol (DTT), 0.1% IGEPAL, 1% plant protease inhibitors (Sigma)), filtered through two layers of Miracloth and subjected to centrifugation at 16 000 × g, 15 min, 4°C. The resulting supernatant was incubated with anti-FLAG Agarose (Sigma) for 2 h at 4°C on a rotating mixer. The agarose resin was then washed twice with lysis buffer and boiled in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. SDS-PAGE was conducted using Tris-glycine gels and proteins were then transferred to nitrocellulose membranes for immunoblotting. Antibodies were diluted in TBST + 5% (w/v) nonfat dried milk as follows: 1:500 anti-NRP(D/E)2, 1:250 anti-RDR2, 1:500 anti-NRP(B/D/E)11 and 1:2000 anti-FLAG-HRP (Sigma). Anti-rabbit-HRP (Santa Cruz Biotechnology) diluted 1:5000 was used as secondary antibody to bind the primary antibodies. Native antibodies to NRP(D/E)2, RDR2 and NRP(B/D/E)11 were previously described (25 (link),31 (link),37 (link)).