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GOGAT | Glutamine oxoglutarate aminotransferase

265 €
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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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AS07 242

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Background

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.

Immunogen

KLH-conjugated synthetic peptide well conserved in known GOGAT sequences from different species including Arabidopsis thaliana Fd-GOGAT 1 Q9ZNZ7, At5g04140 and Fd-GOGAT 2 Q9T0P4, At2g41220

Host Rabbit
Clonality Polyclonal
Clone
Purity Serum
Format Lyophilized
Quantity 50 µl
Reconstitution For reconstitution add 50 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)
Related products
Additional information

This antibody can be used as a plastidial marker

application information
Recommended dilution

1 : 1000 with standard ECL (WB)

Expected | apparent MW

177 | 170-180 kDa depending upon the species

Confirmed reactivity

Arabidopsis thaliana, Miscanthus giganteus, Nicotiana tabacum, Oryza sativa, Phaseolus vulgaris, Populus tremula, Solanum lycopersicum, Spartina sp., Zea mays

Predicted reactivity

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, Medicago trucutula, Microcystis panniformis, Monoraphidium neglectum, Morus notabilis , Nicotiana tabacum, Ostreococcus lucimarinus, Leptolyngbya boryana, Physcomitrella patens subsp. patens,  Porphyra purpurea, Sutterella wadsworthensis, Sulfurihydrogenibium yellowstonense, Veillonella atypica

Not reactive in

No confirmed exceptions from predicted reactivity known in the moment

Additional information

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.

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]
Takabayashi et al. (2016) Direct interaction with ACR11 is necessary for post-transcriptional control of GLU1-encoded ferredoxin-dependent glutamate synthase in leaves. Sci Rep. 2016 Jul 14;6:29668. doi: 10.1038/srep29668.
Yang et al. (2016). Rice Ferredoxin-Dependent Glutamate Synthase Regulates Nitrogen-Carbon Metabolomes and Is Genetically Differentiated between japonica and indica Subspecies. Mol Plant. 2016 Sep 24. pii: S1674-2052(16)30195-2. doi: 10.1016/j.molp.2016.09.004.
Moscatelli et al. (2015). Characterisation of detergent-insoluble membranes in pollen tubes of Nicotiana tabacum (L.). Biol Open. 2015 Feb 20. pii: BIO201410249. doi: 10.1242/bio.201410249.
Podgórska et al. (2013). Long-term ammonium nutrition of Arabidopsis increases the extrachloroplastic NAD(P)H/NAD(P)+ ratio and mitochondrial reactive oxygen species level in leaves but does not impair photosynthetic capacity. Plant Cell Environ. April 10.

Application example

western blot using anti-GOGAT antibodies
20 µg of total protein from (1) Spartina patens total cell extracted with Protein Extration Buffer, PEB (AS08 300), (2) Spartina alterniflora total cell, extracted with PEB, (3) Miscanthus giganteus total cell extracted with PEB, (4) Zea mays total cell extracted with PEB, (5) Phaseolus vulgaris total cell extracted with PEB, (6) Arabidopsis thaliana total cell extracted with PEB, were separated on 4-12% NuPage (Invitrogen) LDS-PAGE and blotted 1h to PVDF. Blots were blocked immediately following transfer in 2% ECL Advance blocking reagent (GE Healthcare) 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, from Abcam) diluted to 1:50 000 in 2% ECL Advance blocking solution for 1h at room temperature with agitation. The blots were washed as above and developed for 5 min with ECL Advance 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).

western blot using anti-GOGAT antibodies on Arabidopsis thaliana

Approximately 40 µg of total protein from Arabidopsis thaliana wild-type (WT) and gls1-103 mutant (M) leaves, the latter of which showed reduced GOGAT levels, was extracted with a protein extraction solution containing 50mM Imidazole/HCl (pH 7.0 at 4ºC), 20% glycerol, 5mM 6-aminocaproic acid, 1mM EDTA. Protein extracts were then separated on 6% SDS-PAGE and blotted overnight at 4ºC to PVDF using the tank transfer method. Blots were blocked with PBS-T containing 5% skim milk for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 20,000 in PBS-T containing 1% (w/v) BSA for 1h at RT 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 PBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from Sigma A9169) diluted to 1:20 000 in PBS-T containing 1% (w/v) BSA for 1h at RT with agitation. The blot was washed as above and developed for 1 min with Western Lightening Plus (Perkin-Elmer) according to the manufacturer's instructions. Exposure time was 5 min.



Courtesy Dr. Atsushi Takabayashi, Institute of Low Temperature Science, Hokkaido University, Japan

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