PsbO | 33 kDa of the oxygen evolving complex (OEC) of PSII (anti-protein)
AS06 142-33 | Clonality: Polyclonal | Host: Rabbit | Reactivity: A.thaliana, C. reinhardtii, C. vulgaris, C. sp. DT, E. crus-galli, F. arundinacea cv. Kord, H. pluvialis, H. spontaneum, H. vulgare, N. benthamiana, P. abies, P. banksiana, P. sativum, P. miliaceum, S. oleracea, S. tuberosum cultivar Taedong Valley, Synechococcus sp. PCC 7942 and PCC6803, T. salsuginea, T.aestivum
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Native purified 33 kDa protein from Spinacia oleracea UniProt: P12359
35 | 33 kDa
1 µg of chlorophyll from Pisum sativum thylakoids and from mesophyll (M) and bundle sheath (BS) thylakoids of Zea mays, Echinochloa crus-galli, Panicum miliaceum extracted with 0.4 M sorbitol, 50 mM Hepes NaOH, pH 7.8, 10 mM NaCl, 5 mM MgCl2 and 2 mM EDTA were loaded to lanes. Samples were denatured with Laemmli buffer at 75 0C for 5 min and were separated on 12% SDS-PAGE and blotted 30 min to PVDF using wet transfer. Blot was blocked with 5% milk for 2h at room temperature (RT) with agitation. Blot was incubated in the primary antibody AS06 142-33 (Lot 1902) at a dilution of 1: 2000 overnight at 40C with agitation in 1% milk in TBS-T. The antibody solution was decanted and the blot was washed 4 times for 5 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG HRP conjugated, from Agrisera, AS09 602, Lot 1902, as recommended) diluted to 1:20 000 in 1 % milk in TBS-T for 1h at RT with agitation. The blot was washed 5 times for 5 min in TBS-T and 2 times for 5 min in TBS, and developed for 1 min with 1.25 mM luminol, 0.198 mM coumaric acid and 0.009% H2O2 in 0.1 M Tris- HCl, pH 8.5. Exposure time in ChemiDoc System was 100 seconds
Courtesy of Dr. Wioleta Wasilewska-Dębowska, Warsaw University, Poland
2 µg of total protein from Arabidopsis thaliana leaf (1), Hordeum vulgare leaf (2), Chlamydomonas reinhardtii total cell (3), Synechococcus sp. 7942 total cell (4), Anabaena sp. total cell (5), all 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 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% blocking solution for 1h at room temperature with agitation. The blots were washed as above and developed for 5 min with chemiluminescent detection reagent according the manufacturers instructions. Images of the blots were obtained using a CCD imager (FluorSMax, Bio-Rad) and Quantity One software (Bio-Rad).
Note: Detection level in algal species can be improved by adjustment of western blot protocol. Please inquire.
Total IgG fraction has been purified by 40% ammonium sulpgate precipitation followed by DEAE cellulose chromatography
This antibody can be used as a loading control for Chlamydomonas reinhardtii while it not so suitable for higher plants as accumulation of these proteins might drop to 12.5-25 % of the WT level in mutants defective for PSII core (Schult et al. 2007).
PSII reaction centre components are generating the redox potential required to drive highly oxidizing water splitting reaction. Four Mn atoms are present on a lumenal surface and form the catalyctic site of the water-splitting reaction which is in close association with the 33 kDa (PsbO), 23 kDa (PsbP) and 17 kDa (PsbQ) extrinistic subunits of oxygen evolving complex OEC. A 33-kDa extrinsic protein is also termed the Mn-stabilizing protein (MSP), however recent evidences shown that it is C-terminal domain of PsbA (D1) protein which is involved in in the assembly and stabilization of the OEC.
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Yang-Er Chen et al. (2017). Responses of photosystem II and antioxidative systems to high light and high temperature co-stress in wheat. J. of Exp. Botany, Volume 135, March 2017, Pages 45–55.