Anti-DCP5 | Protein decapping 5

Samples:
1 - HA-DCP1 (transient in N.benthamiana)
2 - HA-DCP2 (transient in N.benthamiana)
3 - HA-DCP5 (transient in N.benthamiana)

Total protein extracted freshly from 2 leaf discs (Ø6mm) of 4-week-old Nicotiana benthamiana or Arabidopsis thaliana leaves in 150 μL extraction buffer (100 mM Tris pH=7.5, 1mM EDTA, 3% SDS) + 50 μL 4X Laemmli buffer (BioRad) and denatured at 95°C for 10 min. Samples were separated in on 10 % SDS-PAGE and blotted to PVDF membrane (pore size of 0,2 μm), using semi-dry transfer (7 min; 1.3A up to 25V). Blot was blocked with 5 % milk for 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1000 ON/4°C with agitation. The antibody solution was decanted, and the blot was rinsed briefly twice, then washed once for 15 min and 2 times for 5 min in TBS-T at RT with agitation. Blot was incubated in matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1: 10 000 in 5% milk for 1h/RT with agitation. The blot was washed as above, plus one last wash with TBS (without Tween) for 5 min and developed with a following chemiluminescent detection reagent. Exposure time were: 30 seconds, 4 min, 20 seconds, 10 seconds and 2 min (DCP5 HA Nicotiana benthamiana & DCP5 Arabidopsis thaliana, respectively).

Courtesy of Dr. Manuel González Fuente, Faculty of Biology and Biotechnology, Ruhr-University of Bochum, Germany