15 µg of total protein from 4-weeks-old Arabidopsis thaliana plants extracted with Tris-HCl 50 mM pH 7.8, 0.1 mM EDTA, 0.2 % (V/V) Triton X-100; prepared in 0.063 M Tris-HCl buffer, pH 6.8, containing 2 % sodium dodecyl sulfate (SDS; w/v), 10 % glycerol (v/v), 0.006 % bromophenol blue (w/v) and 10 mM DTT; and denatured at 95 °C for 5 min. Proteins were separated on 12 % SDS-PAGE and blotted 1 h to PVDF using semi-dry transfer. Blots were blocked with 3 % (w/v) skimmed milk powder diluted in TBS-T O/N at 4 °C with agitation. Blot was incubated in the primary antibody at a dilution of 1: 2.000 for 1 h at RT with agitation in TBS-T + 3 % (w/v) of skimmed milk powder. The antibody solution was decanted and the blot was washed 3 times for 10 min in TBS-T + 3 % (w/v) of skimmed milk powder at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from Agrisera, Product No: AS09 602 lot 2012) diluted to 1:10.000 in TBS-T + 3 % (w/v) of skimmed milk powder for 1 h at RT with agitation. The blot was washed as above but with TBS and developed for 5 min with chemiluminescent detection reagent. Exposure time was 20 seconds.

Courtesy of Dr. Luisa M. Sandalio, CISIC, Spain