RpoA | RNA polymerase alpha subunit (chloroplast)

no confirmed exceptions from predicted reactivity known in the moment

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