Anti-SymRK I Symbiosis receptor kinase

300 mg of frozen plant tissues from Nicotiana benthamiana transformed with 35S:LjSymRK-GFP or 35S:SlSymRK-GFP were grinded in liquid nitrogen and proteins were extracted freshly with 300 µL of extraction buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 10 mM EDTA, 10% Sucrose, 1% Igepal, 2mM DTT, 1X Protease Inhibitor cocktail. The extract was incubated 30 min with agitation at 4°C, then centrifuged at full speed at 4°C for 10 minutes. Supernatant was collected and mixed with Sample Buffer. The extract was denaturated at 65°C for 5 minutes and loaded on a 10% SDS page. Then, it was blotted with wet transfer 2h 15 minutes at 4°C on Immobilon-FL PVDF, (pore size of 0,45 µm). Blot was blocked with 5% Milk/TBS-T for 1h at RT with agitation. Blot was incubated overnight with primary antibody with dilution of 1:1000 in 1% nonfat milk/TBS-T at 4°C with agitation. The antibody solution was decanted, and the blot was rinsed 3 times 10 minutes at RT with 1XTBS-T with agitation. Blot was incubated at RT for 1h with secondary antibodies anti-Rabbit IRDye 800 1:10000 (LICOR). The membrane was visualized with Odyssey scanner machine at 700 for the Marker and 800 for the antibody signal.

Courtesy of Dr. Katarzyna Parys, LMU Munich, Germany