FtsH1 + FtsH5 | ATP-dependent zinc metalloprotease FtsH1 + FtsH5 (chloroplastic)

Total proteins were isolated from Arabidopsis (Arabidopsis thaliana) wild type (Col) and mutant lacking FtsH2 (yellow variegated2 [var2]). Samples were immediately frozen in liquid nitrogen and pulverized with a microtube homogenizer. Proteins were extracted by adding appropriate extraction buffer. After measurement of chlorophyll concentration, equally loaded supernatants (based on chlorophyll [0.5 µg chlorophyll/lane]) were subjected to SDS-PAGE analysis. Proteins were separated on 12% SDS-PAGE gel and blotted 1h to PVDF membrane. Blots were blocked in 1% BSA in PBST buffer for 1 h at room temperature. Then, blots were incubated in the primary antibody (anti-VAR1) at a dilution of 1:5000 for 1 h. After washing 2 times for 10 min in PBST buffer, blots were incubated in the secondary antibody (anti Rabbit IgG) at a dilution of 1:5000 for 1 h. Blots were washed 2 times for 10 min in PBST buffer. Chemiluminescent detection reagent was used for signal detection. Images of the blots were obtained using ChemiDoc™ XRS (Bio-rad). Exposure time was 2 seconds.
Absence of FtsH2 in var2 mutant results in decreased amount of FtsH1 which together form a hetero-hexamer complex.

Courtesy of Dr. Yusuke Kato, Plant Light Acclimation Research Group, Okayama University, Japan