IDH | Isocitrate dehydrogenase

AS06 203A | Clonality: Polyclonal | Host: Rabbit | Reactivity: A. thaliana, C.annuum, L. esculentum, O. sativa, P. sativum, S. tuberosum, Z. mays | cellular [compartment marker] of mitochondrial matrix

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product information
Background		Plant NADH dependent isocitrate dehydrogenase enzyme is located in mitochondrial matrix. This enzyme is classified as an oxidoreductase and its function is to catalyze a reaction in the citric acid cycle, specifically the sequential dehydrogenation and decarboxylation of isocitrate to form a-ketoglutarate. It removes hydrogens from its substrate, isocitrate. In addition to this process, it functions as a decarboxylase, removing a CO2 from the six-carbon substrate to form a five-carbon product mentioned above as a-ketoglutarate. There are two forms of this enzyme NADP+ and NAD+ dependent.
Immunogen		KLH-conjugated peptide 1 and peptide 2 conserved in all higher plants mitochondrial, NAD dependent isocitrate dehydrogenase subunits including Arabidopsis thaliana IDH-I Q8LFC0, At4g35260 and IDH-II P93032, At2g17130
Host		Rabbit
Clonality		Polyclonal
Clone
Purity		Affinity purified serum
Format		Lyophilized in PBS pH 7.4
Quantity		50 µg
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		AS04 054 \| anti-AOX1/2 rabbit antibody, marker of inner mitochondrial membrane AS04 053A \| anti-COXII rabbit antibody marker of inner mitochondrial membrane AS07 212 \| anti-VDAC1 rabbit antibody marker of outer mitochondrial membrane
Additional information		Peptide used to elicit this antibody is not conserved in NADPH dependent anzymes, partially conserved across eukaryotic Idh subunits. Some conservation across bacterial which contain the NAD-dependent form of Idh (as opposed to the NADP-dependent form).

application information
Recommended dilution		1 : 5 000 (WB)
Expected \| apparent MW		39 \| 45 kDa (Arabidopsis thaliana)
Confirmed reactivity		Arabidopsis thaliana, Brassica oleracea, Capsicum annuum, Lycopersicum chilense, Nicotiana benthamiana, Oryza sativa, Solanum lycopersicum, Pisum sativum, Solanum sogarandium, Solanum tuberosum, Zea mays
Predicted reactivity		Brachypodium distachyon, Brassica napus, Capsella rubella, Citrus sinensis, Glycine max, Hordeum vulgare, Malus x domestica, Medicago truncatula, Nicotiana tabacum, Phaseolus vulgaris, Theobroma cacao, Triticum aestivum, Vitis vinifera, Zea mays
Not reactive in		Chlamydomonas reinhardtii
Additional information		Cellular [compartment marker] of mitochondrial matrix
Selected references		Rurek et al. (2018). Mitochondrial Biogenesis in Diverse Cauliflower Cultivars under Mild and Severe Drought Involves Impaired Coordination of Transcriptomic and Proteomic Response and Regulation of Various Multifunctional Proteins. Preprints 2018, 2018010276 (doi: 10.20944/preprints201801.0276.v1). Fujii et al. (2016). The Restorer-of-fertility-like 2 pentatricopeptide repeat protein and RNase P are required for the processing of mitochondrial orf291 RNA in Arabidopsis. Plant J. 2016 Jun;86(6):504-13. doi: 10.1111/tpj.13185. Yin et al. (2016). Comprehensive Mitochondrial Metabolic Shift during the Critical Node of Seed Ageing in Rice. PLoS One. 2016 Apr 28;11(4):e0148013. doi: 10.1371/journal.pone.0148013. eCollection 2016. Rurek et al. (2015). Biogenesis of mitochondria in cauliflower (Brassica oleracea var. botrytis) curds subjected to temperature stress and recovery involves regulation of the complexome, respiratory chain activity, organellar translation and ultrastructure. Biochim Biophys Acta. 2015 Jan 21. pii: S0005-2728(15)00016-X. doi: 10.1016/j.bbabio.2015.01.005.

Application example

western blot detection using anti-Idh antibodies

20 µg of total protein from (1) Arabidopsis thaliana leaf extract, (2) Arabidopsis thalianafraction enriched with mitochondria, (3) Arabidopsis thaliana pure mitochondria, (4) Pisum sativum pure mitochondria, (5) Solanum tuberosum pure mitochondria were separated on 4-12% SDS-PAGE and blotted to nitrocellulose. Blots were blocked immediately following transfer in 5% milk powder in TBS. Blots were incubated in the primary antibody at a dilution of 1: 5 000 for 1h at room temperature with agitation, followed by an incubation with a secondary antibody and a series of washes. Blots were developed using ECL reagent (GE Healthcare).

* Band detected at ca. 90 kDa is suspected to be a dimmer of Idh, since this band is depleted upon peptide competition experiment.

western blot using anti-plant IDH antibodies

15 µg of total protein stem extract from Lycopersicum esculentum (1), pure mitochondrial fraction isolated from stems of Lycopersicum esculentum (2), pure mitochondrial fraction isolated from stems of Capsicum annuum (3), pure mitochondrial fraction isolated from tubers of Solanum tuberosum (4) were separated on 10% SDS-PAGE and blotted onto nitrocellulose . After blocking with 5% milk in TBST , blots were incubated with the primary antibody at a dilution of 1:1000 in TBST for 1.5h at room temperature. Following incubation and wash steps, blots were incubated with secondary Anti-Rabbit IgG , Alkaline Phosphatase Conjugate for 1 hour at a dilution of 1:40000 . Blots were developed with the alkaline phosphatase detection system using NBT/BCIP (SIGMA).

Courtesy of Bartosz Szabala, Institute of Plant Genetics, Polish Academy of Science.

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