Anti-GUN1 | Pentatricopeptide repeat-containing protein GUN1, chloroplastic

Samples:
1. 50 ug of 36-h-old WT seedlings (Arabidopsis thaliana)
2. 50 ug of 36-h-old gun1 mutant (Arabidopsis thaliana)
3 and 4: 50 ug of two 36-h-old independent overexpression lines with GFP tag in gun1 background (Arabidopsis thaliana)

50 µg/well of total protein extracted freshly from 36-h-old Arabidopsis thaliana seedling. Exact buffer components were: 0.0625 M Tris-HCl (pH 6.8), 1% (w/v) SDS, 10% (v/v) glycerol, 0.01% (v/v) 2-mercaptoethanol. and denatured with exact buffer components at 95 °C/5min.  Samples were separated on 10% SDS-PAGE at room temperature (RT) and blotted for 1.5 h to Immune-Blot PVDF ( Bio-Rad (0.2 um)) using: wet in the cold. Blot was blocked with 5 % milk for: 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1:  500 ON/4°C with agitation. Then washed once for 20 min and 4 times for 5 min in TBS-T at RT with agitation. Blot was incubated in matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1: 10 000 for 1h/RT with agitation. The blot was washed as above and developed with a following chemiluminescent detection reagent. Exposure time was 5 seconds.

Note regarding background bands: the aim of the protocol which was used above was to confirm that GUN1 protein is detected. Background signal can be decreased by adjustment of Western blot protocol: primary antibody incubation at 1: 1000 1h/RT.