Anti-DCP1 | mRNA-decapping enzyme subunit

1) Col-0 (Arabidopsis thaliana leaf)

2) YFP-DCP1, mock-treated (Arabidopsis thaliana leaf)

3) YFP-DCP1, Pst-infected (Arabidopsis thaliana leaf)

4) YFP-DCP1/atg5-1, mock-treated (Arabidopsis thaliana leaf)

5) YFP-DCP1/atg5-1, Pst-infected (Arabidopsis thaliana leaf)

DCP1-RFP (transient expression in Nicotiana benthamiana)

Total protein extracted freshly from 2 leaf discs (Ø6 mm) of 4-week-old Arabidopsis thaliana or Nicotiana benthamiana leaves in 150 μL extraction buffer (100 mM Tris pH=7.5, 1mM EDTA, 3% SDS) + 50 μL 4X Laemmli buffer and denatured at 95°C for 10 min. Samples were separated in on 10 % SDS-PAGE, total protein was visualized via Stain Free imaging 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: 2000 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 chemiluminescent detection reagent. Exposure times were: 5, 13, 0.5 and 5 seconds (DCP1 and GFP in Arabidopsis thaliana, and DCP1 and RFP in Nicotiana bentamiana, respectively).

Courtesy of Dr. Manuel Gonzalez Fuente Ruhr-Universität Bochum, Germany