WUS | WUSCHEL protein

Plant material: Col-0 wild type (1), pWUS::WUS-GFP (2), expression level comparable to wild type pUbiquitin::mCherry-GR-WUS (3), over-expresses the wuschel protein and also has ectopic expression. Used as a positive control for the westerns. 200mg of 5-7 day old seedling were extracted with 600 μl of 1x SDS-PAGE loading buffer (50mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 1% β-MeSH, 12.5mM EDTA, 0.02% bromophenol blue) supplemented with 1% Sigma Protease Inhibitor Cocktail (Cat. Num: P9599). Protein amounts in the extracts were determined by amidoblack. 80 μg (lane 1 and lane 2) or 20 μg (lane 3) of total protein was separated on 10 % SDS-PAGE and blotted for 1.5h to PVDF using semi-dry transfer. Blots were blocked with 4% Milk and 1% BSA in 1x TBST for 1h at room temperature (RT) with agitation. The blot was incubated in the primary antibody at a dilution of 1:1 000 in 1x TBST (0.1% Tween-20) with 1% BSA for 16h at 4°C with agitation. The antibody solution was decanted and the blot was rinsed briefly, then washed 3x for 10 min in 1x TBST at RT with agitation. Blot was incubated in secondary antibody diluted to 1:5 000 in 1x TBST for 1h at RT with agitation. The blot was washed as above and developed for 2 min with ECL (Advansta, K-12045-D20) according to the manufacturer's instructions. Exposure time was 120 seconds.

Courtesy of Matyas Medzihradszky, Centre for Organismal Studies, University of Heidelberg, Germany