GOGAT | Glutamine oxoglutarate aminotransferase
AS07 242 | Clonality: Polyclonal | Host: Rabbit | Reactivity: A. thaliana, G. max, N. tabacum, O. sativa, Populus sp., S. lycopersicum, S. elongatus, S. oleracea , Z. mays | Plastidial marker
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177 | 170-180 kDa depending upon the species
Arthrospira platensis PCC 7345, Beta vulgaris, Brachypodium distachyon, Burkholderia glumae, Camellia sinensis, Chloroflexi bacterium, Chlamydonas reinhardtii, Gracilaria tenustipitata, Cyanobacteria, Crocosphaera watsonii WH 8502, Cyanidioschyzon merolae (strain 10D), Galdieria sulphuraria , Glycine max, Helicosporidium sp. ATCC 50920, Hydrogenobaculum sp. HO, Lactococcus lactis subsp. lactis bv. diacetylactis str. TIFN2, Leptospira interrogans serovar Bataviae str. HAI135, Leptolyngbya boryana, Medicago trucutula, Microcystis panniformis, Monoraphidium neglectum, Morus notabilis, Nannochloropsis gaditana, Nicotiana tabacum, Ostreococcus lucimarinus,Physcomitrella patens subsp. patens, Porphyra purpurea, Sutterella wadsworthensis, Sulfurihydrogenibium yellowstonense, Veillonella atypica
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20 µg of total protein cell extract extracted with Protein Extration Buffer, PEB (AS08 300) from leafs of (1) Spartina patens (2) Spartina alterniflora l, (3) Miscanthus giganteus , (4) Zea mays, (5) Phaseolus vulgaris, (6) Arabidopsis thaliana 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: 10 000 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-rabbit IgG horse radish 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 according the manufacturers instructions. Images of the blots were obtained using a CCD imager (FluorSMax, Bio-Rad) and Quantity One software (Bio-Rad).
This antibody can be used as a plastidial marker for leaf material, not roots where levels of GOGAT are too low.
A 40 kDa band present in A. thaliana sample is not competed away during antibody neutralization assay. In this assay free peptide used for antibody production is incubated together with anti-GOGAT antibodies. Due to the MW of this protein we suggest to use a gradient gel for protein separation and a longer transfer time.
Glutamine oxoglutarate aminotransferase (abbreviated as GOGAT) is an enzyme involved in synthesis of glutamate from glutamine and alpha-ketoglutarate. GOGAT has two forms in plants: ferredoxin-dependent GOGAT (Fd-GOGAT) and NADH-dependent GOGAT (NADH-GOGAT). 95% of GOGAT found in plants is the Fd-GOGAT type. Fd-GOGAT is encoded by two genes, glu1 and glu2 found on chromosomes 5 and 2 respectively (in Arabidopsis). Fd-GOGAT (both forms) is highly conserved among plants, red algae, and cyanobacteria.
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