Anti-RpoC1 | RNA polymerase beta' subunit (chloroplast)

Samples:

5 µg/well of total protein extracted from Arabidopsis thaliana and Zea mays in 100 mM Tris-HCL pH7.5, 10%glycerol, 5mM EDTA and 2MM EGTA buffer and denatured in 66mM Tris-HCL pH6.8, 0.2% glycerol, 0.23% SDS, 600mM BME at 50°C/10 min. Samples were separated on 4-20 % SDS-PAGE Novex™ Tris-Glycine Mini Protein Gels (4–20%, 1.0 mm thick) and blotted overnight to nitrocellulose (0.22um), using: wet transfer. Blot was blocked with 5 % milk for: 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1:500 for 2h/RT with agitation in 5% non-fat milk TBS-T with agitation. The antibody solution was decanted, and the blot was rinsed briefly 4 times in TBS-T for 5min each with agitation. Blot was incubated in matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:1000 in 5% non-fat milk TBS-T for 1h/RT with agitation. The blot was washed as above and developed with a following chemiluminescent detection reagent AgriseraECL SuperBright. Exposure time was 5 minutes.

Courtesy of Jie Shen and Dr. Alice Barkan, University of Oregon, USA

Stroma, thylakoid, nuclei and total protein from Arabidopsis thaliana and Zea mays were extracted with 50 mM HEPES-KOH pH 8, 330 mM sorbitol exaction buffer. Components were separated on 12% SDS-PAGE and blotted overnight to Nitrocellulose membrane using wet transfer. Blots were blocked with 5% non-fat milk for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 500 for 2h at RT 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 TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:10 000 in for 1h at RT with agitation. The blot was washed as above and developed for 5min with chemiluminescent detection reagent of extreme low femtogram range. Exposure time was 120 seconds.

Courtesy of Jie Shen and Dr. Alice Barkan, University of Oregon, USA