Anti-Cyt f | Cytochrome f protein (PetA) of thylakoid Cyt b6/f-complex (algal)

5-25 µg/well of total protein extracted freshly from Chlamydomonas reinhardtii.  Exact buffer components were at 4 °C in 150 µL of lysis buffer containing 7 M urea, 2 M thiourea, 18 mM Tris-HCl, 14 mM Trizma base, 4% (w/v) CHAPS and 0.2% (v/v) Triton X-100, to which 20 µL of protease inhibitor cocktail (Complete Mini, Roche Diagnostics), dissolved in water as per the manufacturer’s instructions, were added and denatured with 3 µL of 1 M dithiothreitol (DTT) for 30 min at 4 °C before centrifugation (20 000 g, 10 min, 4 °C) and supernatant with 2x loading buffer heated at 85°C for 5 min before loading.  Samples were separated at RT on 12 % SDS-PAGE and blotted for 1 h to nitrocellulose (pore size of  0.45 µm), using:  semi-dry transfer at RT. Blot was blocked with 5 % milk for 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1:  10 000 for 1h/RT with agitation in TBS-T. The antibody solution was decanted and the blot was rinsed briefly twice, then washed 3 times for 10 min in TBS-T at RT with agitation. Blot was incubated in  matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, AS09 602, Agrisera) diluted to 1: 25 000 in  for 1h/RT with agitation. The blot was washed as above and developed with a following chemiluminescent detection reagent.  Exposure time was 20 seconds.

Courtesy of Dr. Thomas Roach, University of Innsbruck, Austria