application example

2.5 and 5 µg of total leaf of Arabidopsis thaliana extracted with 25 mM HEPES pH 7.5 and 10 mM MgCl2 were separated on 12% SDS-PAGE and blotted 1h to PVDF. Blots were blocked with dry milk for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 10 000 overnight 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 TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, AS09 602 from Agrisera ) diluted to 1:10 000 in for 1h at RT with agitation. The blot was washed as above and developed for 5 min with ECL according to the manufacturers instructions. Exposure time was 3 minutes. Pre-immune -signal obtained with pre-immune serum. Affinity purified - signal obtained with BAK1 affinity purified antibodies. Peptide competition - signal obtained after saturation of anti-BAK1 antibody with BAK1 peptide used for this antibody production.

Courtesy of Dr. Andrea Trotta, University of Turku, Finland.