Anti-L32 | 50S ribosomal protein L32 (chloroplastic)

Samples:
1 - 10 ug of Nicotiana tabacum wild-type whole leaf extract
2 - 10 ug of Nicotiana tabacum Rpl32 over-expresser whole leaf extract, contain native chloroplast Rpl32 (6.3 kDa) and fusion protein Rpl32-YFP (33.2 kDa)
3 – 10 ug of Nicotiana tabacum wild-type root extract, used as negative control that does not contain chloroplast proteins
ladder: MW markers

Tobacco leaf and root protein was extracted freshly from Nicotiana tabacum (Exaction buffer components were: 0.7 M sucrose, 0.5 M Tris, 50 mM EDTA, 0.1 M KCL, 2% β-Mercaptoethanol, 2% Complete proteases inhibitor), mixed thoroughly and then added same volume of Phenol, precipitated in 0.1 M NH4OAc in Methanol, and resuspended in 10% SDS. Then 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/10 min. 10 µg/well of protein samples were separated in the cold on 12% SDS-PAGE and blotted for 20 V/overnight to nitrocellulose (pore size of 0.2 um), using: wet transfer in the cold. Blot was blocked with 4 % milk for 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1000 for ON/4°C with agitation in TBS-T. The antibody solution was decanted, and the blot was rinsed briefly twice, then washed 3 times for 10 min in TBS-T at RT with agitation. Blot was incubated in matching secondary antibody (Agrisera anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1: 10000 in TBS-T for 1h/RT with agitation. The blot was washed as above and developed with a following chemiluminescent detection reagents. Exposure time was 30 seconds.

Courtesy of phd student. Jinghan Liu and Dr. Reimo Zoschke, Max Planck Institute, Germany