NR | Nitrate reductase, assimilatory

AS08 310 | Clonality: Polyclonal | Host: Rabbit | Reactivity: A. thaliana, H. vulgare, C. reinhardtii, red alga Gracilaria gracilis, diatom Thalassiosira sp., P. tricornutum Bohlin, P. yunnanensis Dode, S. lycopersicum, S. tuberosum

Product Information

Immunogen

KLH-conjugated synthetic peptide derived from conserved domain in NADH-NR protein sequences including A.thaliana NR1 P11832, At1g77760 and NR2 P11035, At1g37130

Host Rabbit

Clonality Polyclonal

Purity Affinity purified serum in PBS, pH 7.4

Format Lyophilized

Quantity 50 µg

Reconstitution For reconstitution add 25 µl of sterile water.

Storage Store lyophilized/reconstituted at -20°C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles. Please, remember to spin tubes briefly prior to opening them to avoid any losses that might occur from lyophilized material adhering to the cap or sides of the tubes.

Tested applications Western blot (WB)

Recommended dilution 1 : 500 -1 : 1000 (WB)

Expected | apparent MW

103 kDa | 117 kDa

Reactivity

Confirmed reactivity Arabidopsis thaliana, Chlamydomonas reinardtii, Cucumis sativus, red alga Gracilaria gracilis, Hordeum vulgare, Phaeodactylum tricornutum Bohlin accession Pt1 8.6, Populus yunanensis Dode, Solanum lycopersicum, Solanum tuberosum, Thalassiosira sp. (diatom), Vigna radiata

Predicted reactivity Arabis alpina, Brachypodium distachyon, Brassica napus, Brassica rapa subsp. pekinensis, Capsella rubella, Citrus clementina, Citrus sinensis, Chlorella vulgaris, Dunaliella salina, marine Diatoms, Coffea canephora, Eucalyptus grandis, Glycine max, Glycine soja, Gossypium arboretum, Helianthus annuus, Lycopersicum esculentum, Morus alba, Nicotiana tabacum, Nicotiana attenuata, Nicotiana benthamiana, Oryza sativa, Phaseolus vulgaris, Phytophthora infestans, Physcomitrella patens, Prunus persica, Ricinus communis, Sorghum bicolor, Spinacia oleracea, Solanum lycopersicum, Symbiodinium microadriaticum, Theobroma cacao, Zea mays, Vitis vinifera

Not reactive in

Aspergilus niger, Emiliania huxleyi, Tisochrysis lutea

Application examples

Application examples Application example

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 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 AS09 602, 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 chemiluminescent detection reagent pf extreme femtogram range, 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.

Additional information

ECL based detection systems are adviced to use since to low signal intensity can be obtaied with BCIP/NBT system.

In Chlamydmonas reinhardtii anti-NR antibody is also reacting with L-Aminoacid Oxidase (a nitrogen scavenging enzyme induced during nitrogen starvation).

Related products

AS12 2611 | NRT1.1 | NITRATE TRANSPORTER 1.1, rabbit antibody
AS12 2612 | NRT2.1 | NITRATE TRANSPORTER 2.1, rabbit antibody
AS09 473 | NRT1.4 | nitrate transporter, rabbit antibody

Plant protein extraction buffer

Secondary antibodies

Background

Assimilatory nitrate reductase (NR), (EC.1.6.6.1) 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).

Product citations

Selected references Jayawardena et al. (2016). Elevated CO2 plus chronic warming reduces nitrogen uptake and levels or activities of nitrogen -uptake and -assimilatory proteins in tomato roots. Physiol Plant. 2016 Nov 28. doi: 10.1111/ppl.12532. [Epub ahead of print]
Chen et al. (2016). The role of nitric oxide signalling in response to salt stress in Chlamydomonas reinhardtii. Planta. 2016 Sep;244(3):651-69. doi: 10.1007/s00425-016-2528-0. Epub 2016 Apr 26.
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.
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