PFOR | pyruvate oxidoreductase

Chlamydomonas reinhardtii total cell extract 15 µg/lane from cells acclimated to dark anoxia following  (1) 2 h; (2) 4h; (3) 8 h of anoxia imposition. Total proteins were extracted with 50 mM Tris buffer (pH 8.0) containing 10 mM EDTA and 2% SDS,  and were separated by SDS-PAGE using a 10% polyacrylamide gel  and then transferred 1h at RT to PVDF membranes. The membranes were blocked with a 5% milk in TBS-T for 2 h. Blot was incubated in the primary anti-PFOR antibody at a dilution of 1: 1 000 over night at 4°C with agitation in 3 % milk. 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  for 1h at RT with agitation. The blot was washed as above and developed for 5 min with chemiluminescence detection reagent, according to the manufacturers instructions. Exposure time was 5 seconds.

Courtesty Dr. Caludia Catalanotti, Carnegie Institution, USA