Protein Extraction From N. benthamiana Leaves

Infected leaves were collected 10 days post-infiltration, snap frozen in liquid nitrogen and ground using a mortar and a pestle. One gram of ground-up leaves was resuspended in 3 mL of the extraction buffer (50 mM Tris, 150 mM NaCl, 1 mM DTT, 0.1% Triton X-100, pH 7.5), containing a cocktail of protease inhibitors (Cat. No. 11873580001, Roche, Basel, Switzerland). Proteins were extracted by vigorously vortexing the ground-leaf suspensions at 4°C. Additional incubation of the total leaf suspensions overnight at −20°C helped with cell lysis. Thawed suspensions were cleared by centrifugation at 14,000 × g for 30 min at 4°C. Total soluble and insoluble fractions containing rAra h 2 were prepared as follows. An aliquot from the homogenized leaf suspension was taken for mixing with the reducing sample buffer (200 mM Tris, 300 mM DTT, 4% SDS, 40% glycerine, bromphenol blue), heated at 95°C and loaded as the total fraction. The rest of the homogenized leaf suspension was centrifuged at 14,000 × g for 15 min. An aliquot from the supernatant was mixed with the reducing sample buffer, heated at 95°C and loaded as the soluble fraction. The pellet was mixed with the reducing sample buffer, and an aliquot was taken for heating at 95°C prior to loading.