application example

50 µg of total protein from Arabidopsis thaliana nuclei extracted with Nuclei Lysis Buffer (1% SDS, 50mM Tris-HCl, 10mM EDTA, Complete EDTA-free) were separated on 12 % SDS-PAGE and blotted 1h to PVDF membrane (WestranS 0,20 µm, Whatman).HD2C+/- heterozygote nuclear extract (1), HD2C -/- homozygote nuclear extract (2), HD2C -/- homozygote nuclear extract (3), HD2C -/- homozygote nuclear extract (4), 35S:HD2C-GFP nuclear extract (5), 35S: HD2C-GFP nuclear extract (6), Arabidopsis thaliana Col-0 nuclear extract (7), Arabidopsis thaliana Col-0 nuclear extract (8). Blots were blocked with 5% fat free milk in TBST for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 2 500 in 2,5% fat-free milk in TBST overnight at 40C with agitation. The antibody solution was decanted and the blot was washed with TBST three times for 10 min and blocked in 10% fat-free milk in TBST for 10 min. Next, blot was incubated in secondary antibody diluted to 1:5 000 in 5% fat-free milk in TBST for 2h at RT with agitation. The blot was washed six times for 10 min with TBST and developed for 5 min with ECL+ (Amersham) according to the manufacturers instructions. Exposure time was 30 seconds.

Note: Additional band in lanes 5 and 6 comes from HD2C-GFP.

Courtesy Daniel Buszewicz, University of Warsaw, Poland