Idh | isocitrate dehydrogenase

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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

antibody format

rabbit

polyclonal

affinity purified serum, in PBS pH 7.4

lyophilized

quantity

180 µg

for reconstitution add 180 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)

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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 with standard ECL (WB)
expected \| apparent MW		39 \| 45 kDa (Arabidopsis thaliana)
confirmed reactivity		Arabidopsis thaliana, Capsicum annuum, Lycopersicum esculentum, Pisum sativum, Solanum tuberosum, Zea mays
predicted reactivity		dicots including Brassica napus, Vitis vinifera, monocots including Oryza sativa, Zea mays
not reactive in		Chlamydomonas reinhardtii
additional information		cellular [compartment marker] of mitochondrial matrix
selected references		Lee et al. (2012). Mitochondrial Targeting of the Arabidopsis F1-ATPase γ-Subunit via Multiple Compensatory and Synergistic Presequence Motifs. Plant Cell, dec. 18.

application example

western blot detection using anti-Idh antibodies

20 µg of total protein from (1) Arabidopsis thaliana leaf extract, (2) Arabidopsis thaliana fraction 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. 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 SIGMA 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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