Anti-TIM10| Mitochondrial import inner membrane translocase

Samples: 
1 - 50 ug of Arabidopsis thaliana seedling isolated mitochondria extract #1​
2 - 50 ug of Arabidopsis thaliana seedling isolated ​ mitochondria extract #2​
3 - 50 ug of Arabidopsis thaliana cell culture isolated mitochondria extract​
4 - 50 ug of Pisum sativum seedling isolated mitochondria extract​ 5 - 50 ug of Oryza sativa seedling isolated mitochondria extract​ Mark: Blue Protein Standard MW markers​


50 µg/well of total protein extracted freshly from the seedlings of Arabidopsis thaliana, Pisum sativum and Oryza sativa.  The mitochondria were extracted according to Murcha and Whelan (2015) mitochondria isolation protocol pages 1-12. Samples were separated in the cold on 14 % SDS-PAGE  and blotted for 1 h to nitrocellulose (pore size of  0.45 um), using semi-dry method in the cold. Blot was blocked with 10 % skim milk for: 4°C/ON with agitation. Blot was incubated in the primary antibody at a dilution of 1: 500 in 1X TBS-Tween for ON/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 matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1: 10 000 in 1X TBS-Tween for 1h/RT with agitation. The blot was washed as above and developed with a following chemiluminescent detection reagent. Exposure time was 10 minutes.
Mitochondria were extracted according to the method described here:  MURCHA, M. W. & WHELAN, J. 2015. Isolation of Intact Mitochondria from the Model Plant Species Arabidopsis thaliana and Oryza sativa. In: WHELAN, J. & MURCHA, M. W. (eds.) Plant Mitochondria: Methods and Protocols. New York, NY: Springer New York.


Courtesy of Valencia Marisa, The University of Western Australia, Australia