NdhH | NAD(P)H-quinone oxidoreductase subunit H (chloroplastic)
AS16 4065 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana, Zea mays
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|Expected | apparent MW||
45 | 49 kDa
|Confirmed reactivity||Zea mays|
|Predicted reactivity||Arabidopsis thaliana, Cannabis sativa, Hordeum vulgare|
|Not reactive in|
|Additional information||This product can be sold containing proclin if requested.|
|Selected references||Nikkanen et al. (2018). Regulation of chloroplast NADH dehydrogenase-like complex by NADPH-dependent thioredoxin system. CSH, BioRixiv, doi: https://doi.org/10.1101/261560.|
Total proteins of Arabidopsis thaliana leaf were precipitated with 10% TCA, washed with acetone and solubilized in a buffer with 8M urea, 100 mM Tris-HCL pH 7.5, 1 mM EDTA, 2% SDS. 10 ug of TCA precipites were separated in a BioRad TGX 4-20% precast gels and blotted 1h to a PVDF membrane using Höefer semi-dry blotter. Blot was blocked with 8 % milk in TTBS for 30 min. at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 5 000 overnight in +4°C. The antibody solution was decanted and the blot was washed 3 x 5 min with TTBS at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from Agrisera, AS09 602) diluted to 1:20 000 in 1% milk/TTBS for 2h at RT with agitation. The blot was washed 3x5 min in TTBS and 1x5 min in TBS at RT with agitation and developed for 5 min with ECL according to the manufacturer's instructions.
Courtesy Msc. Lauri Nikkanen, University of Turku, Finland
5µg of total protein from Zea mays leaf seedling tissue was extracted with protein extraction buffer (100 mM Tris pH7.5, 10% glycerol, 1 mM EDTA, 1mM EGTA, 2mM pMSF, 0.002 mg leupeptin, 0.002 mg pepstatin A, 2.5 mM DTT) and denatured in 1XPSB (33 mM Tris pH 6.8, 50 mM 2-Mercaptoethanol, 2% SDS, 10% glycerol, 0.1% bromophenol blue) at 70°C for 5 min, separated on 4-20% Tris-Glycine SDS-PAGE and blotted overnight to nitrocellulose using tank transfer. Blots were blocked with 4% milk in 1XTBSt for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1:500 for 1h at RT with agitation in 4% milk in 1XTBS-T. The antibody solution was decanted and the blot was rinsed briefly twice, then washed 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, AS09 602, Agrisera) diluted to 1:10 000 in 4% milk in 1XTBS-T for 1h at RT with agitation. The blot was washed as above and developed for 2 min with chemiluminescent detection reagent.
Courtesy Rosalind Carrier, University of Oregon, USA
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