GlnA | Glutamine synthetase
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KLH-conjugated synthetic peptide derived from available bacterial GlnA sequences with perfect conservation in alpha, beta, gamma Proteobacteria, Enterobacteria, Thermotogales, Low GC Gram+, Cyanobacteria (except weak conservation with Trichodesmium thiebautii) including Synechocystis PCC 6803 Q59981
Alpha, beta, gamma proteobacteria, Arthropsira sp. PCC 8005, Crenarchaeotes, Enterobacteria, Escherichia coli, Euryarchaeotes, Thermotogales
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3 µg of total protein from Trichodesmium IMS 101 extracted with Agrisera Protein Extration Buffer (AS08 300) (1-8) and GlnA protein standard 0.3, 0.15, 0.07 pmol (9-11) were separated on 4-12% NuPage (Invitrogen) LDS-PAGE and blotted 1h to PVDF. Blots were blocked immediately following transfer in 2% blocking reagent 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: 50 000 for 1h at room temperature 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 room temperature with agitation. Blots were incubated in secondary antibody (anti-hen IgY horseradish peroxidase conjugated) diluted to 1:50 000 in 2% blocking solution for 1h at room temperature with agitation. The blots were washed as above and developed for 5 min with chemiluminescent detection reagent in extreme femtogram range, according the manufacturer's instructions. Images of the blots were obtained using a CCD imager nd Quantity One software Exposure time was 10 seconds.
Glutamine synthetase (EC=126.96.36.199) is the key enzyme in the incorporation of mineral nitrogen into glutamine. Activity of this enzyme is controlled by adenylarion under conditions of abundant glutamine.
Brown et al. (2008). Flux capacities and acclimation costs in Trichodesmium from the Gulf of Mexico. Marine Biol. 154:413-422.
Burns et al. (2006). Inorganic carbon repletion constrains steady-state light acclimation in the cyanobacterium Synechococcus elongatus. J. Phycol. 42:610-621.
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