application example

Arabidopsis thaliana wild ecotype Columbia was grown for four weeks under continuous illumination and then transferred to complete darkness for five days. Several leaves were harvested from the plants before they were transferred to darkness (0 d) or after they were kept for five days (5 d). Protein was extracted with the SDS extraction solution containing 50 mM Tris (pH 6.8), 10% (w/v) glycerol, 2% (w/v) SDS and 6% (v/v) 2-mercaptoethanol. Protein extract equivalent to 1 mg leaf material was loaded and separated on 14% SDS-PAGE and blotted 1h to PVDF. Blots were blocked with PBS-T containing 1.5% skim milk for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 10 000 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 PBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from GE Healthcare ) diluted to 1:20 000 in for 1h at RT with agitation. The blot was washed as above and developed for 1 min with ECLplus according to the manufacturers instructions. Exposure time was 5 min.