PGR5 | Proton gradient regulation 5
AS16 3985 | clonality: polyclonal | host: rabbit | reactivity: Arabidopsis thaliana, Picea abies, Pinus sylvestris, Solanum lycopersicum
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1: 1000 - 1: 5000 (WB)
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Thylakoids were isolated according to Sirpiö et al (2011, Methods Mol Biol. 2011; 775:19-30). 3 µg/µl of total chlorophyll from Arabidopsis thaliana col-0 (1) and Arabidopsis thaliana pgr5 mutant (Munekage et al. 2002, Cell) (2) were denatured in Laemmli buffer at room temperature (15 min) were separated on 12 % SDS-PAGE mini-gels with 6 M urea and blotted for 1h to a PVDF membrane using a semi-dry transfer. Membranes were blocked with 5% milk in TBS for 1h at room temperature and incubated in antisera (1:1000 in 1 % milk/TTBS) overnight at 4°C with slow agitation, then in secondary antibody (anti-rabbit IgG HRP conjugated, AS09 602, Agrisera) 1:20 000 in 1% milk/TTBS) for 2 hours at RT with slow agitation. Blots were detected for 5 min with a chemiluminescent substrate. Exposure time was 2 min.
The pgr5 mutant has a point mutation that affects the function of the PGR5 protein, therefore a band is still seen in the western blot.
Courtesy Dr. Peter Gollan, University of Turku, Finland
Rantala et al. (2020). PGR5 and NDH-1 systems do not function as protective electron acceptors but mitigate the consequences of PSI inhibition. Biochim Biophys Acta Bioenerg. 2020 Jan 11;1861(3):148154. doi: 10.1016/j.bbabio.2020.148154.
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