ACO1 | Aconitase
AS09 521 | Clonality:Polyclonal | Host:Rabbit | Reactivity: Arabidopsis thaliana, Solanum lycopersicum
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Arabidopsis ACO1 (AT4G35830, Q42560), codon 120 – 898 (C-terminus), was cloned in fusion with a N-terminal 6xHis tag, and over-expressed in E. coli. All recombinant protein accumulated in inclusion bodies, which were purified by centrifugation and solubilised in 6 M guanidine-HCl. The protein was refolded by dilution in 100 mM Tris-HCl 8.5, 10% (v/v) glycerol, 2 mM dithiothreitol, and concentrated prior to immunisation.
98 kDa. Note that ACO1, ACO2 and ACO3 cannot be distinguished in size by standard SDS-PAGE.
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Chlamydomonas reinhardii, cyanobacteria
Western blot analysis of 1) 5 ng purified 6xHis-AtACO1 (Δ119, 87 kDa); 2) 2 ng 6xHis-AtACO1; 3) Total protein (15 μg) from Arabidopsis thaliana leaves were extracted with 2 volumes 50 mM Tris-HCl pH 8.0, 5% (v/v) glycerol, 1% (w/v) sodium dodecyl sulphate, 10 mM NaEDTA, 1 mM phenylmethanesulfonyl fluoride; 4) 15 µg of purified mitochondria from Arabidopsis thaliana cell culture, 5) 15 µg of protein from Arabidopsis thaliana chloroplasts
Proteins were separated by SDS-PAGE and blotted onto nitrocellulose (Whatman Protran BA 83, 0.2 μm). Blots were blocked in Tris-buffered saline (TBS) with 0.1% (v/v) Tween 20 and 5% (w/v) dried skimmed milk for 1 h at room temperature, and incubated with anti-AtACO1 antibodies diluted 1:10,000 in fresh block solution (10 mL per 8 x 6 cm blot) for 2 h at room temperature. The blot was washed 3 times with block solution, then incubated with horse-radish peroxidase conjugated anti-rabbit IgG antibodies, diluted 1: 5,000 in block solution, for 45 minutes. The blot was washed 2 times with block solution and 2 times with TBS-Tween. The signal was developed with standard ECL reagents and Kodak X-Omat LS film.
Note: as visible in lane 2, detection of recombinant AtACO1 falls below 2 ng of recombinant protein.
Arabidopsis expresses three highly similar aconitase isozymes (ACO1/ AT4G35830, ACO2/AT4G26970 and ACO3/AT2G05710), of which ACO1 is the cytosolic isoform, while ACO2 and ACO3 are predominantly located in the mitochondria (Arnaud et al 2007, Bernard et al 2009). The combined abundance and activity of the mitochondrial aconitases is about 3 times higher than the cytosolic pool (Bernard et al 2009).The Arabidopsis isoforms are more similar in amino acid sequence to mammalian iron-regulatory proteins (IRP-1) than to the mammalian and yeast mitochondrial aconitases.
The antibody recognises all three Arabidopsis aconitase isoforms (ACO1, ACO2 and ACO3, see Bernard et al 2009). Possible differences in affinity have not been precisely quantified. Sensitivity threshold is between 2 and 10 ng for WB / ECL (see figure). Antibodies will recognize aconitase isoforms in denaturing and native gel electrophoresis.
Aconitase is a single subunit enzyme of the tricarboxylic acid cycle (or Krebs cycle) in the mitochondria. A cytosolic isoform is also part of the glyoxylate cycle. Aconitase catalyzes the dehydration / hydration of citrate to iso-citrate, via cis-aconitate as an intermediate. The reaction is facilitated by an iron-sulphur cluster in the active site of the enzyme. The iron-sulphur cluster is somewhat unstable, especially under oxidative stress, and loss of the cofactor leads to degradation of the protein.Alternative names: ACO, citrate hydro-lyase 1,2,3
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