EPYC1 | Essential Pyrenoid Component 1

25 µg/well of total protein extracted freshly from Chlamydomonas reinhardtii cells with 0.1mM NaOH + Protease inhibitor cocktail set IV (1:200; 539136, Sigma) and denatured with Sample buffer (1.6 mM EDTA, 1.6% SDS, 40 mM DTT, 8% Glycerol, 0.016% Bromophenol Blue, 333 mM Tris Base) at 70°C for 5 min, were separated on 12% SDS-PAGE and blotted 1h to nitrocellulose membrane (pore size of 0.2 um), using semi-dry transfer. Blot was blocked with 5% milk for 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1 000 in TBS-T 5% milk 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 Agrisera matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, AS09 602) diluted to 1:25 000 in TBS-T 5% milk for 1h/RT with agitation. The blot was washed as above and developed for 5 min with Agrisera ECL Bright. Exposure time was 10 seconds.
Courtesy of Dr. Justin Findinier, Carnegie Institution for Science, USA