RPS15 | 30S ribosomal protein S15, chloroplastic

5 µg/well of total protein extracted freshly from Nicotiana tabacum RPS15 mutant (1) and wildtype (2) with 0.7 M sucrose, 0.5 M Tris, 50 mM EDTA, 0.1 M KCl, 2% β-Mercaptoethanol, 2% Complete proteases inhibitor, and Phenol, and denatured with 1 vol of 2x protein loading buffer (125 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 25 mM EDTA, 0.4 mg/ml Bromophenol blue, 2% β-Mercaptoethanol) at 64°C for 10 min. Samples were separated on 12 % SDS-PAGE and blotted 20V/ON or 350A/2h at 4°C to nitrocellulose (pore size of 0.2 µm), using wet transfer. Blot was blocked with 4% milk for 30min/RT on a gentle rocking. Blot was incubated in the primary antibody at a dilution of 1: 1 000 for ON/4°C on a gentle rocking in TBS-T. The antibody solution was decanted, and the blot was rinsed briefly with TBS-T, then washed three times for 10min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (Goat Anti-Rabbit IgG (H + L)-HRP Conjugated) diluted to 1:10 000 in 2% dry milk, 15 mM EDTA and TBS-T for 1h/RT with agitation. The blot was washed as above and developed with chemiluminescent detection reagent following manufacture's recommendations. Exposure time was 1.5 min.

Courtesy of Drs Jinghan Liu and Reimo Zoschke, Max Planck Institute of Molecular Plant Physiology, Germany