Anti-PsbS | 22 kDa Lhc-like PSII protein (rabbit antibody)

5 µg of total extract from (1) Arabidopsis thaliana leaf,  (2) Spinacia oleracea  (3) Hordeum vulgare (4) Zea mays extracted with PEB (AS08 300) were separated on 4-12% NuPage (Invitrogen) LDS-PAGE and blotted 1h to PVDF. Blots were blocked immediately following transfer in 2% ECL Advance blocking reagent (GE Healthcare) in 20 mM Tris, 137 mM sodium chloride pH 7.6 with 0.1% (v/v) Tween-20 (TBS-T) for 1h at room temperature with agitation. Blots were incubated in the primary antibody at a dilution of 1: 10 000 for 1h at room temperature 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 room temperature with agitation. Blots were incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:50 000 in 2% ECL Advance blocking solution for 1h at room temperature with agitation. The blots were washed as above and developed for 5 min with ECL Advance detection reagent according to the manufacturers instructions. Images of the blots were obtained using a CCD imager (FluorSMax, Bio-Rad) and Quantity One software (Bio-Rad). Exposure time was 30 seconds. 

Courtesy of Dr. Laura Roy, University of Amsterdam, The Netherlands

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10 µL of total protein extracted freshly from Arabidopsis thaliana (A), Brachypodium distachyon (B) and Oryza sativa (R) with 1X Laemmli Buffer (63 mM Tris-HCl pH 6.8, 10% (w/v) glycerol, 2% SDS and Bromophenol Blue q.b./1 pick) diluted to 1:2 and denatured with 25 mM DTT at 95ºC for 4 min, were resolved on a 10 % SDS-PAGE and blotted 1h to PVDF membrane (pore size of 0.45 μm, GE Healthcare), using wet transfer. Blot was blocked with Blocking solution containing 5% milk in TBS-T, for 1h/RT with agitation. Membrane was incubated with the primary antibody at a dilution of 1:2000 in Blocking solution containing 5% milk in TBS-T, for 1h/RT with agitation in TBS-T. The antibody solution was removed, and the membrane was washed 3 times for 10 min in TBS-T at RT with agitation. The membrane was then incubated in the secondary antibody (anti-rabbit IgG, horse radish peroxidase conjugated) diluted to 1:20 000 in Blocking solution containing 5% milk in TBS-T, for 1h/RT with agitation. After incubation with the secondary antibody, membrane was washed 6 times for 5 min in TBS-T at RT with agitation and developed for 1 min with Agrisera ECLSuperBright. Exposure time was 10 seconds.

Courtesy of Dr. Nelson Saibo,Plant Gene Regulation Lab (GPlantS Unit), ITQB NOVA, Portugal

The 22 kDa PsbS protein of photosystem II functions in the regulation of photosynthetic light harvesting. Along with a low thylakoid lumen pH and the presence of de-epoxidized xanthophylls, PsbS is necessary for photoprotective thermal dissipation of excess absorbed light energy in plants, measured as non-photochemical quenching of chlorophyll fluorescence. Synonymes: NPQ4 (NONPHOTOCHEMICAL QUENCHING).