application example

80 µg of total protein from Arabidopsis thaliana wt Columbia (right lane) and Arabidopsis thaliana phot1phot2 double mutant (left lane) were separated on 9% SDS-PAGE and blotted 2h to PVDF. Blots were blocked immediately following transfer in PBS-T 5% milk for 1h at room temperature with agitation. Blots were incubated in the primary antibody at a dilution of 1: 5000 overnight at 4^oC with agitation. The antibody solution was decanted and the blot was rinsed briefly, then washed 3 times for 5 min in PBS-T at room temperature with agitation. Blots were incubated in secondary antibody (anti-IgG horse radish peroxidase conjugated, from Agrisera AS09 602) diluted to 1:10 000 for 1h at room temperature with agitation. The blots were washed as above and developed with WestPico detection reagent (PIERCE) according to the manufacturers instructions. Exposure time was 300 seconds.