The 20 μg of soluble proteins from 3-week old Solanum tuberosum (1) and Arabidopsis thaliana (2), one-week old Lupinus angustifolius (3), Pisum sativum (4) and Zea mays L. (5) leaves extracted with buffer containing 50 мM Hepes-KOH, pH 7.5, 330 мM sorbitol, 2 мM EDTA, 1 мM MgCl2, 5 мM ascorbate, 0.05% BSA were mixed with sample buffer and denatured for 5 min at 70°C. Samples were separated on 10% SDS-PAGE and blotted 1h to nitrocellulose membrane (Amersham Protran) using tank wet-transfer (Bio-Rad) in standard transfer buffer in presence of 20% methanol. Transfer of proteins to the membrane was checked using 1% Ponceau S staining before the blocking step. Blots were blocked in buffer (5% low-fat milk in 1xPBS, 0.1% Tween-20) for 1h at room temperature (RT) with agitation. Blots were incubated in the primary antibody at a dilution of 1:2 000 for 1 h at RT with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, and then washed once for 15 min and 3 times for 5 min in PBS-T at RT with agitation. Blot was incubated in secondary antibody (goat anti-rabbit IgG, AS09 602, Agrisera) diluted to 1:30 000 (Agrisera) in for 1h at RT with agitation. The blot was washed as above and developed for 5 min with Clarity Western ECL Substrate and ChemiDoc detection system (Bio-Rad). Exposure time was 60 seconds.
Courtesy Dr. Elena Pojidaeva, Laboratory of Plant Gene Expression, Timiryazev Institute of Plant Physiology RAS, 127276 Moscow Russia
0.3 µg of total protein from Synechococcus sp. PCC7942 (1) and 20 µg of leaf soluble proteins from 3-week old Arabidopsis thaliana leaves (2) were separated on 3-8 % Tris-acetate NuPage gel (invitrogen) and blotted 1,5 h to supported introcellulose. Blots were blocked for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 2 000 in 1xTBS-T for 1h at RT with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from Agrisera, AS09 602) diluted to 1:75 000 in for 1h at RT with agitation. The blot was washed as above and developed for 5 min with ECL according to the manufacturers instructions. Exposure time was 120 seconds.
Courtesy of Dr. A. Clarke, Göteborg University, Sweden
