PHR1 - Phosphate starvation response 1 (monocots)

Samples: purified MBP tag protein, 50 ng, (1), purified MBP-HvTF (MYB-family like PHR1) tag protein 50 ng, (2), purified MBP+HvPHR1, 50 ng, (3).  Samples were separated on 13% SDS-PAGE and blotted 1h to PVDF/nitrocellulose using semi-dry transfer. Blot was blocked with 10% milk for 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1 000 for 1h/RT with agitation in TBS-T. The antibody solution was decanted and the blot was rinsed briefly twice, then washed three times for 10 min in TBS-T at RT with agitation. Blot was incubated in Agrisera matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated AS09 602) diluted to 1:50 000 in 10% milk with TBS-T for 1h/RT with agitation. The blot was washed as above and developed for 3 min with chemiluminescent detection reagent. Exposure time was 3 minutes.

Courtesy of Msc. Paweł Sega, Institute of Molecular Biology and Biotechnology, Department of Gene Expression, UAM, Poznań, Poland