ATG9 | Autophagy-related protein 9 (N-terminal)

Increasing amount of microsomal fraction of Arabidopsis thaliana cell culture (2,3,4).  Microsomal protein was extracted accoring to Maudoux et al. (2000).   and denatured with loading buffer (2 % SDS) at 37°C for 1h before SDS-PAGE gel electrophoresis and blotted to PVDF using semi-dry. Blots were blocked with 3 % nonfat milk + 0.1 % goat serum in TBS-T (Tween 20)  for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 10 000 + 1 % goat serum for ON at 4°C 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 TBS-T + 1 % goat serum for 1h at RT with agitation. The blot was washed as above and developed with chemiluminescent detection reagent. Exposure time was  seconds.
Red marker band has apparent mass of 72 kDa. 

Courtesy of Dr. Henri Batoko, University of Louvain, Belgium