NdhS | NAD(P)H-quinone oxidoreductase subunit S (chloroplastic)
AS16 4066 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana, Hordeum vulgare
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1:10 000 (WB)
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no confirmed exceptions from predicted reactivity known in the moment
Arabidopsis thaliana leaves from wildtype and ndho mutant were frozen in liquid nitrogen. Leaf tissue was homogenized in ice-cold isolation buffer (330 mm Suc, 25 mm HEPES-KOH, pH 7.4, 10 mm MgCl2, and 10 mm NaF) and filtered through Miracloth. The filtrate was centrifuged at 6,000g for 5 min at 4°C. The thylakoid pellet was resuspended in 25 mm HEPES-KOH, pH 7.4, 10 mm MgCl2, and 10 mm NaF, centrifuged at 6,000g, for 5 min at 4°C, and finally suspended in the isolation buffer and stored at −80°C. The chlorophyll content of isolated thylakoids was determined according to Porra et al. (1989)
Samples were denatured with Laemmli buffer at 65°C for 5 min. and were separated on 12 % SDS-PAGE and blotted 1h to PVDF (0.45um), using semi-dry transfer. Blot was blocked with 5 % milk 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 10000 in TBS-T ON/4°C with agitation. The antibody solution was decanted and the blot washed once for 15 min and 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in Agrisera matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:25 000 in for 1h/RT with agitation. The blot was washed as above and incubated for 5min min with chemiluminescent detection reagents. Exposure time was 5-10 min.
Courtesy Dr. Lauri Nikkanen, University of Turku, Finland
NAD(P)H-quinone oxidoreductase subunit 2 is involved in the transfer of electrons from NAD(P)H:plastoquinone, via FMN and iron-sulfur centers, to quinones in the photosynthetic chain and possibly in a chloroplast respiratory chain. Alternative names: NAD(P)H dehydrogenase, subunit 2, NADH-plastoquinone oxidoreductase subunit 2.
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