AOX2 | Plant alternative oxidase 2

Samples:
Total protein 1-4
Cytoplasmic fraction
5-8 Nuclear fraction 9-12

20 µg/well of total protein extracted freshly from fractionation of Arabidopsis thaliana 7-day-old green seedlings and denatured with 2x Laemmli Sample Buffer at 90°C for 5 min were separated on 12 % SDS-PAGE  and blotted 1h to nitrocellulose (pore size of  0.45  um), 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: 1 000 for ON/4°C with agitation in TBS-T. 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 Agrisera matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:25 000 in  for 1h/RT with agitation. The blot was washed as above and developed for 5  min with Agrisera ECLBright. Exposure time was 200 seconds.

Courtesy of Dr. Zhengyao Shao, The University of Texas at Austin , USA