DRB4 | Double-stranded RNA-binding protein 4

 Application example


100 µg of total protein from Arabidopsis thaliana wt, drb4, and 35S-DRB4  leaves extracted with isolation buffer (50 mM Tris-HCl, pH 7.5, 10% glycerol, 150 mM NaCl, 10 mM MgCl₂, 5 mM EDTA, 5 mM DTT, and 1 X protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO) and denatured by boiling at 100°C. The proteins were separated on 10% SDS-PAGE and blotted 1.5 hrs to PVDF membrane using wet transfer. Blots were blocked with 5% milk powder  for 30 min at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1,000 for 60 min at RT with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed 3 times for 10 min in phosphate buffer (containing 0.1% Tween 20) at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG alkaline phosphatase conjugated) diluted to 1:10,000 for 120 min at RT with agitation. The blot was washed as above and developed with  NBT/BCIP (Fisher Chemicals) using Alkaline phosphatase detection system. The DRB4 specific band was seen within 20 mins and showed a better signal when allowed to develop for longer duration (overnight in this case).

Courtesy of Dr. Gah-hyun Lim and Prof. Pradeep Kachroo, University of Kentucky, Lexington, USA

300 µg of total protein from Arabidopsis thaliana Col-0 and drb4 mutant flowers, extracted according to Hurkman and Tanaka (Plant Physiol. 1986 Jul; 81(3):802-6) and denatured with WB loading buffer at 95°C for 5 min, were separated on 15% SDS-PAGE and blotted 90 min to PVDF membrane using wet transfer. Blots were blocked with 5% milk powder in PBS, Tween 20 0.1% for 30 min at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1:5000 overnight at 4°C with agitation. The antibody solution was decanted and the blot was washed 4 times for 10 min in PBS Tween20 0.1% at RT with agitation. Blot was incubated in secondary antibody (goat anti-rabbit IgG horse radish peroxidase conjugated, provided by Agrisera) diluted to 1:25000 in PBS 1x, Tween 20 0.1%, milk powder 5% for 3h30 at RT with agitation. The blot was washed as above, incubated for 3 min with Lumi-Light^PLUS Western Blotting Substrate (Roche) and revealed using X-ray films. Exposure time was 5 seconds.

Courtesy of Dr. Patrice Dunoyer, Institut de Biologie Moléculaire des Plantes du CNRS (IBMP-CNRS), France