RpoA | RNA polymerase alpha subunit (chloroplast)

Stromal fraction proteins (100 µg) were extracted from Arabidopsis thaliana cell culture grown under dark (7-d-D) and 150 μmol m-2s -1 constant white light for 7 days (7-d-L). Proteins were separated by NativePAGE 3-12% Bis-Tris Gel and detected using anti-RpoB antibody. PEP complex separated by BN-PAGE were further resolved by 4-12% NuPAGE and blotted 250mA for 1.5h to 0.45 µm PVDF using wet transfer. Blot was blocked with 5% milk for 1h/RT with agitation. Blot was incubated in the primary antibody anti-RpoA at a dilution of 1: 500 for ON/4°C with agitation in TBS-T. The antibody solution was decanted and the blot was rinsed briefly three times, then washed once for 10 min and repeat 3 times in TBS-T at RT with agitation. Blot was incubated in Agrisera matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated; AS09 602) diluted to 1:10 000 for 1h/RT with agitation. The blot was washed as above and developed with AgriseraECL SuperBright (AS16 ECL-S) for 12 sec for dark sample and 2 sec for light sample.

Courtesy Yan Ji, PhD student Åsa Strand Group Department of Plant Physiology Umeå Plant Science Centre (UPSC), Sweden