Application example

100 µg of total protein from Arabidopsis thaliana wild-type (1) and transgenic plants expressing GUS under HRT promoter (2) were extracted with write buffer containing  50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM MgCl₂, 5 mM EDTA, 10% glycerol, 0.1% NP-40, 0.1% protease inhibitor cocktail . The proteins were denatured by boiling for 5 minwere separated on 10% SDS-PAGE  and blotted 30 min to PVDF using tank transfer. Blots were blocked with  TBST containing 5% non-fat dry milk   for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 10 000 for 2h at RT with agitation in TBS-T. The antibody solution was decanted and the blot was rinsed briefly twice, then washed 3 times for 15 min each in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:10 000 in  for 1h at RT with agitation. The blot was washed as above and developed with  Clarity MAX (Bio-RAD). Exposure time was 10 seconds.