application example

Ten days old seedlings of Oryza sativa (cv Nipponbare) were submerged under water for 3 days, epigeal biomass was collected from control and treated samples for protein extraction. 25 µg of total protein were extracted with SDS Extraction Buffer (60mM Tris-HCl pH 8.0, 2 % SDS, 1,5 % Sucrose). Proteins were separated on 4-12 % SDS-PAGE (Criterion XT Precast Gel, Biorad) and blotted 7 min to PVDF with Trans-Blot Turbo Transferase System (Biorad). Blot was blocked with TTBS-milk for 1 h at RT with shaking. Blot was incubated in the primary antibody at a dilution of 1:5 000 overnight at 4 °C with shaking. The blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TTBS at RT with shaking. Blot was incubated in secondary antibody (goat anti-rabbit IgG HRP conjugated, AS09 602 Agrisera) diluted 1:20 000 in TTBS-milk for 45 min at RT with shaking. The blot was washed as above and developed for 5 min with ECL (LiteAblot Turbo, Euroclone) according to the manufacturers instructions. Total exposure time was 3 min. Images of the blot were obtained using BioSpectrum AC Imaging System (UVP). Arrow marks Sub1C protein.

Courtesy of Dr. Viet-The Ho, Scuola Superiore Sant'Anna, Italy