RDR6 | RNA-dependent RNA polymerase 6

Nicotiana tabacum, Solanum lycopersicum, Zea mays

50 µg of total protein from Arabidopsis thaliana whole vegetative rosette wild type Col-0 (a) rdr6-12 mutant (b) extracted with extraction buffer (50 mM Tris pH 7.5; 150 mM NaCl; 1 mM EDTA; 10 % v/v Glycerin; 1 mM DTT, 1x Complete Protease Inhibitor Cocktail, Roche) and denatured with Laemmli buffer at 95°C/5 min., were separated on 7.5 % SDS-PAGE and blotted 1.5 h to PVDF using tank transfer. Blots were blocked with blocking buffer (5% milk powder; 1x TBS; 0.1% Tween-20) overnight at 4°C with agitation. Blot was incubated in the primary antibody at a dilution of 1:6000 for 2h at RT with agitation. The antibody solution was decanted and the blot was rinsed briefly and then washed tree times for 15 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from Agrisera, AS09 602) diluted to 1:20 000 in blocking buffer for 1h at RT with agitation. The blot was washed as above and developed for 5 min with chemiluminescent detection reagent and expose to Amersham Hyperfilms ECL for 3 minutes. 

Courtesy of Dr. Pablo Manavella, Instituto de Agrobiotecnología del Litoral (IAL), Argentina

RDR6 (RNA-dependent RNA polymerase 6) is involved in trans-acting siRNA and other siRNA biogenesis and required for post-transcriptional gene silencing and natural virus resistance.

Alternative names:Protein silencing defective 1, Protein supressor of gene silencing 2, RNA.directed RNSA polymerase 6.