NR | Nitrate reductase, assimilatory (100 ĩl)
AS08 310-100 | Clonality: Polyclonal | Host: Rabbit | Reactivity: A. thaliana, H. vulgare, Ch. reinhardtii, red alga Gracilaria gracilis, diatom Thalassiosira sp. , P. tricornutum Bohlin, P. yunnanensis Dode, S. tuberosum
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103 kDa | 117 kDa
20 µg of total protein from Arabidopsis thaliana leaf (1) and Hordeum vulgare leaf (2) were extracted with Protein Extraction Buffer PEB (AS08 300). Samples were diluted with 1X sample buffer (NuPAGE LDS sample buffer (Invitrogen) supplemented with 50 mM DTT and heat at 70°C for 5 min and keept on ice before loading. Protein samples were separated on 4-12% Bolt Plus gels, LDS-PAGE and blotted for 70 minutes to PVDF using tank transfer. Blots were blocked immediately following transfer in 2% blocking reagent (GE RPN 2125; Healthcare) or 5% non-fat milk dissolved 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: 5 000 (in blocking reagent) for 1h at room temperature with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, and then washed 1x15 min and 3x5 min with TBS-T at room temperature with agitation. Blots were incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, recommended secondary antibody AS10 1489, Agrisera) diluted to 1:20 000 in blocking reagent for 1h at room temperature with agitation. The blots were washed as above. The blot was developed for 5 min with TMA-6 (Lumigen) detection reagent according the manufacturers instructions. Images of the blots were obtained using a CCD imager (VersaDoc MP 4000) and Quantity One software (Bio-Rad). Exposure time was 5 minutes.
ECL based detection systems are adviced to use since to low signal intensity can be obtaied with BCIP/NBT system.
For working with diatom samples ECL Advance (GE Healthcare) or other more sensitive ECL detection reagent is recommended.
In Chlamydmonas reinhardtii anti-NR antibody is also reacting with L-Aminoacid Oxidase (a nitrogen scavenging enzyme induced during nitrogen starvation).
Assimilatory nitrate reductase (NR), (EC.18.104.22.168) catalyses the reduction of nitrate to nitrite in the cytoplasm. Plants contain 2 forms of NR: NADH-NR (most common form in plants and algae, predominantly found in green tissues) and NAD(P)H-NR (uses NADH or NADPH as the electron donor, constitutively expressed in plants at a low level). NADH-NR is a homodimer of two identical subunits (100-115 kDa each, hold together by a Mo-cofactor) each of them coded by up to three genes (NR1-3, NIA1-NIA3).
Cheng et al. (2015). Quantitative proteomics analysis reveals that S-nitrosoglutathione reductase (GSNOR) and nitric oxide signaling enhance poplar defense against chilling stress. Planta. 2015 Aug 2.
Johnson and Lecomte (2015). Characterization of the truncated hemoglobin THB1 from protein extracts of Chlamydomonas reinhardtii. v1; ref status: indexed,
Zhang et al. (2014). Heterologous expression of AtPAP2 in transgenic potato influences carbon metabolism and tuber development. FEBS Lett. 2014 Aug 27. pii: S0014-5793(14)00621-8. doi: 10.1016/j.febslet.2014.08.019.
Beyzaei et al. (2014). Response of Nitrate Reductase to Exogenous Application of 5-Aminolevulinic Acid in Barley Plants. J. Plant Growth Regulation, April 2014.
Frada et al. (2013). Quantum requirements for growth and fatty acid biosynthesis in the marine diatom Phaeodactylum tricornutum (Bacilloriophyceae) in nitrogen replete and limited conditions. J. Phycology. Diatom growth and lipid efficiency