Anti-PsbO antibodies, product number: AS06 142-33, detect a PsbO protein in a wide range of different species. Recently, reactivity on a new species, Eucalyptus grandis, has been experimentally confirmed using the following protocol: 13.5 µg/well of total protein extracted freshly from Eucalyptus sp. leaves. Exact buffer components were: 50 mM HEPES NaOH pH 8.0, 5mM DTT, 15 mM NaHCO3, 20 mM MgCl2, 2 mM EDTA, 4% (v/v) EDTA- free protease inhibitor cocktail (Roche Diagnostics), 4% PVPP (w/v), 20% glycerol (v/v), 1:1 Tris-buffered phenol (v/v) and 100 mM CH3COONa in methanol. Extracts were denatured with SDS beta-Mercaptoethanol sample buffer for 10 min at 90°C. Samples were separated at RT on 12% SDS-PAGE and blotted for 1 h to PVDF, using wet transfer at room temperature (RT). Blot was blocked with 5% non-fat milk for 1 h at RT with agitation. Blot was incubated in the primary antibody at a dilution of 1:5000 for 1 h at RT with agitation in TBS-T. The antibody solution was decanted, and the blot was rinsed briefly twice, then washed three times for 10 min with TBS-T at RT with agitation. Blot was incubated in matching secondary antibody (anti-rabbit IgG HRP, Agrisera AS09 602) diluted to 1: 10000 in TBS-T for 1 h at RT with agitation. The blot was washed as above and developed with a following chemiluminescent detection reagent. Exposure time was 12 min. | ![]() Samples: 1: 13.5 µg of Eucalyptus grandis whole leaf extract clone one (g1) |
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