HRP | Horseradish peroxidase

Proteins were isolated from poplar T89, barley, Arabidopsis thaliana, horseradish root mutants were solubilized with 3X LB (6 M urea, 12% SDS, 30% glycerol, 100 mM DTT, 150 mM Tris pH7.0, 0.8% Comassie G-250). 45 µg of total proteins and 45-7.5 µg of HRP were loaded into each lane and separated on 12% SDS-PAGE, and then blotted overnight onto PVDF membrane. Membranes were blocked with milk powder for 2 h and then incubated in the primary antibody solution overnight, which was then decanted and the membrane was washed 3 times for 5 min in TBST. Membrane was incubated at RT for 1 hour in 1:10 000 goat anti-Rabbit secondary antibody AS09 602, followed by washing steps as above. Membrane was developed for 2 min with chemiluminescent detection reagent according to the manufacturer’s instructions and recorded using FujiFilm CCD camera with 10 s increment time for around 100 s.