Anti-Rps2 | Ribosomal subunit 2 (cytoplasmic, dicot)

15 µg/well of total protein extracted freshly from Arabidopsis thaliana 8-day-old seedlings. Exact buffer components were: 4 M Urea buffer components at 4 °C. Samples were separated in the RT on 12 % SDS-PAGE and blotted for 2 h to PVDF (pore size of 0.2 µm), using: we transfer in the cold. Blot was blocked with 10 % milk for 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 3000 for 1h/RT with agitation in PBS with agitation. 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 PBS at RT with agitation. Blot was incubated in matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1: 5000 in for 1 h/RT with agitation. The blot was washed as above and developed with a following chemiluminescent detection reagent: SuperSignal West Pico PLUS (Thermo Fisher). Exposure time was 1 minute.

Note: Faster migration of Rps2 is attributed to presence of 4 M urea in the sample. In the presence of 6 M urea less SDS is going to bind to the protein, and as an effect of that the protein is going to migrate faster than calculated from the sequence.

Courtesy of Dr. Kuo-En Chen, Department of Biology, Washington University in St. Louis, USA