Anti-RNS3 | Ribonuclease 3

Samples:
M - MW markers
1 - 20 µg of Arabidopsis thaliana whole leaf extract
2 - 20 µg of Arabidopsis thaliana mutant, Δ rns3

20 µg/well of total protein extracted freshly from Arabidopsis thaliana rosette leaves.  Exact buffer components were: 150 mM NaCl, 50 mM Tris-HCl pH 7.5, 0.1% NP-40, 1% Protease inhibitor cocktail, 1% DPDS; and denatured with loading buffer (5% 2-Mercaptoethanol, 60 mM Tris-HCl pH 6.8, 2% SDS, 10 % Glycerol, 0.001 % bromophenol blue) at 95°C / 5 min.  Samples were separated in the cold on   4-20 % SDS-PAGE  and blotted for 1 h to nitrocellulose (pore size of 0.45 µm), using: wet transfer in the cold. 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 in TBS-T. The antibody solution was decanted, and the blot was rinsed briefly twice, then washed once for 15 min and 3 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 in TBS-T for 1h/RT with agitation. The blot was washed as above and developed with a following chemiluminescent detection reagent. Exposure time was 2 minutes.

Note: Background signal can be optimized by primary antibody incubation 1h/RT and blocking with 5 % non-fat milk ON/4°C. 

Courtesy of Dr. Lucía Borniego, Innes Lab, Indiana University Bloomington, USA