PsbE | Cytochrome b559 subunit alpha (Algal)

Samples:
1 - 10 μg of Synechocystis PCC 6803 isolated thylakoid membranes
2 - 20 μg of Synechocystis PCC 6803 isolated thylakoid membranes
3 - 10 μg of PsbE deletion mutant Synechocystis PCC 6803 isolated thylakoid membranes

10 or 20 µg/well of total protein from isolated thylakoid membranes with MES buffer (25mM MES/NaOH, pH 6.5, 10 mM MgCl2, 10 mM CaCl2, and 25% glycerol) and denatured with SDS-solubilizing buffer stock (5 % SDS in 25 mM Tris-HCl, pH 7, 1M sucrose) with working concentrations 1% SDS and 2% DTT at 23°C for 1 hour were separated on 16 - 20 % AA, 7 M urea SDS-PAGE and blotted 1h to PVDF (pore size of 40 μm), using wet transfer. Blot was blocked with 0.2 % Tween in TBS for 30 min/RT with agitation. Blot was incubated in the PsbE primary antibody at a dilution of 1: 1 000 overnight at 4°C with agitation in TBS-T. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 5 min in TBS-T at RT with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 5 min in TBS-T at RT with agitation. Blot was incubated in Agrisera secondary antibody (AS09 602) diluted to 1:25 000 in TBS-T for 1h/RT with agitation. The blot was washed as above, incubated for 5 min in AgriseraECL Bright (AS16 ECL-N) and developed in LAS-4000 camera (Fujifim, Japan). Exposure time was 1 minutes.

Courtesy Dr. Josef Komenda, ALGATECH, Czech Republic