NdhF | NAD(P)H dehydrogenase subunit 5 (chloroplastic)
AS13 2711 | Clonality: Polyclonal | Host: Rabbit | Reactivity:
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83 | 58 kDa
Total protein from Oryza sativa rice (CV. 9311) flag leaf at the tillering stage was ground into a fine powder in liquid nitrogen. An 800 ul aliquot of extraction buffer [62.5 mM TRIS-HCl (pH 7.4), 10% glycerol, 0.1% SDS, 2 mM EDTA, 1 mM phenylmethylsulphonyl fluoride (PMSF), 5% (v/v) b-mercaptoethanol] was added to each 300 mg powder sample. The mixture was vortexed and then chilled on ice for 10 min. Samples were centrifuged at 12,000 rpm for 10min at 4 ℃, and the supernatant was collected and stored at –70 ℃. The protein concentrations of the rice samples were determined using the Bradford method (Bradford, 1976). 20 µg of protein was separated on 12 % SDS-PAGE and blotted 1h to PVDF.
Blots were blocked with for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1 000 for 1h at RT 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 RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:10 000 in for 1h at RT with agitation. The blot was washed as above and developed for 5 min with ECL according to the manufacturer’s instructions. Exposure time was 120 seconds.
NDH (NAD(P)H-quinone oxidoreductase subunit 5, chloroplastic) 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-quinone oxidoreductase subunit 5, chloroplastic, NADH-plastoquinone oxidoreductase subunit 5.
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