GLN1 GLN2 | GS1 GS2 glutamine synthetase global antibody
AS08 295 | Clonality: Polyclonal | Host: Rabbit | Reactivity: [global antibody] | A.thaliana, E.tef, G.gracilis (red algae), G. lemaneiformis, M.truncatula, P.patens, P. strobus, S.oleracea, S.lycopersicum, Z.mays
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39-40 kDa (GLN1,cytoplasmic form) , 44-45 kDa (GLN2, chloroplastic form)
Brachypodium distachyon, Brassica napus, Camellia sinensis, Citrus clementina, Cucumis melo, Daphnia magna, Datisca glomerata, Emiliania huxleyi, Eucalyptus grandis, Gazania splendens, Genlisea aurea, Glycine max, Helianthus annuus, Hordeum vulgare, Oryza sativa, Panax quinquefolius, Phaseolus angularis, Phytophthora cinnamomi, Populus trichocarpa, Saccharum officinarum, Securigera parviflora, Solanum lycopersicum, Solanum tuberosum, Stevia rebaudiana, Theobroma cacao, Triticum aestivum, Zea mays, Vitis labrusca
GLN1 dicots including: Brassica napus, Phaseolus vulgaris, monocots including: Hordeum vulgare, Oryza sativa, trees: Pinus sylvestris, Populus sp., Zosteria marina
GLN2 dicots including: Brassica napus, Glycine max, Phaseolus vulgaris, monocots including: Triticum aestivum, Oryza sativa
GLN3: Zea mays,
GLN1 in algae: Chlamydomonas reinhardii
10 µg of total protein extracted freshly from Arabidopsis thaliana wt leaf tissue (Atn non-senescent leaves), Arabidopsis thaliana wt leaf tissue (Ats senescent leaves), Pinus strobus needle tissue (PSA-J) with 1 M Tris-HCl, pH 6.8, 10 % SDS, 15 % sucrose, 0.5 DTT and denatured at 75°C for 5 min. were separated on 10 % Bis-Tris Nupage Novex gel (120 V/45 min. using MES buffer system) and blotted 30 min. to PVDF. Blot was blocked with 5 % non-fat milk 45 min./RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 10 000 for 1h/RT with agitation in TBS with 2 % non-fat milk or ON/4°C with agitation. The antibody solution was decanted and the blot was rinsed briefly twice for 10 min. in TBS at RT with agitation. Blot was incubated in Agrisera matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, AS09 602) diluted to 1:75 000 in for 1h/RT with agitation. The blot was washed as above and developed using chemiluminescent detection. Exposure time was 26.5 seconds.
Courtesy of Dr. Christine Yao-Yun Chang and the Ensminger lab, University of Toronto, Canada
The detection of GS1 and GS2 proteins was performed using the crude extract of soluble proteins from Oryza sativa plants: Ref: Reference (control); D: Drought; CO2: High CO2 D+CO2: Drought + High CO2. Fresh leaves samples were ground until obtaining a fine powder in presence of liquid N2, ice-cold 100 mM K-phosphate buffer (pH 7.0) containing 1 mM EDTA and 2 mM ascorbic acid. After centrifugation at 14,000 x g for 30 min, the supernatant was collected and used as protein extract. All extraction stages were carried out at 4°C. The total soluble protein was measured according to the Bradford’s method. Leaf protein extracts were first separated by SDS-PAGE (Laemmli 1970). Equal amounts of protein (20 µg) were electrophoretically transferred to a nitrocellulose membrane (Towbin et al. 1979). Polypeptide detection was performed using specific polyclonal antibodies against GS1 and GS2 (AS08 295, Agrisera, Sweden). Membranes were blocked for 3 hours with 5% non-fat milk in saline Tris-HCl buffer (100 mM Tris-HCl, pH 7.6, 150 mM NaCl), incubated with GS antibody overnight and after with alkaline phosphatase-conjugated secondary antibody by 6 hours. The protein detection was developed using NBT/BCIP (Sigma-Aldrich©, USA) by adding 1 tablet to 10 mL dH2O, until bands were visualized.
Courtesy of Dr. Ana Karla Lobo, Laboratory of Plant Metabolism, Federal University of Ceara, Brazil
The antibody will recognize both, cytoplasmic and chloroplastic forms of the GS enzyme.
Glutamine synthetase (GLN or GS) is one of the key enzymes involved in nitrogen metabolism of plants. It catalyses the synthesis of glutamine from glutamate and ammonia in an ATP-dependent reaction. There are two general classes of glutamine synthetase in plants: GLN1, a cytosolic form and GLN2, a chloroplastic form. GLN1 is highly abundant in the vascular elements of roots nodules, flowers and fruits, functioning in the assimilation of ammonium and the biosynthesis of glutamine for nitrogen transport. GLN2 is encoded by a single gene and is highly abundant in leaf mesophyll chloroplasts. Here GLN functions in the assimilation of ammonia produced from photorespiration and the reduction of nitrate in the chloroplasts
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