APX1 | Ascorbate peroxidase (algal)

25 µg of total protein from Chlamydomonas reinhardtii extracted with 2 % SDS / 50 mM TRIS pH 6.8 + protease inhibitor cocktail were separated on 12 % SDS-PAGE and blotted for 1 h to PVDF using semi-dry transfer. Blots were blocked with 5 % low-fat milk powder TBS + 0.1 % Tween for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 2,500 for 1 h at RT with agitation. The antibody solution was decanted and the blot was rinsed, then washed 3 times each for 5 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from Sigma) diluted to 1:25,000 in 2 % low-fat milk powder TBS + 0.1 % Tween for 1h at RT with agitation. The blot was washed as above and developed with chemiluminescent detection reagent, according to the manufacturer's instructions. Exposure time was typically 30 seconds.

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