ACO1 | Aconitase
AS09 521 | clonality:polyclonal | host:rabbit | reactivity: Arabidopsis thaliana, Solanum lycopersicum
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1:5 000 – 1:10 000 with standard ECL (WB). At higher concentrations the antibody binds aspecifically resulting in non-specific signals ≤ 60 kDa, including Rubisco subunits.
|Expected | apparent MW||
98 kDa. Note that ACO1, ACO2 and ACO3 cannot be distinguished in size by standard SDS-PAGE.
|Confirmed reactivity||Arabidopsis thaliana ACO1,ACO2 and ACO3 isoforms, Solanum lycopersicum|
dicots including:Cucurbita maxima, Nicotiana tabacum, Ricinus communis, Solanum tuberosum, Vitis vinifera,monocots: Oryza sativa, Zea mays, trees: Picea sitchensis, Populus trichocarpa
|Not reactive in||
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.
|Selected references||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.
Setién et al. (2014). Root phosphoenolpyruvate carboxylase and NAD-malic enzymes activity increase the ammonium-assimilating capacity in tomato. J Plant Physiol. 171:49-63.
Birke et al. (2012). Cysteine biosynthesis, in concert with a novel mechanism, contributes to sulfide detoxification in mitochondria of Arabidopsis thaliana. Biochem J. May 2, ahead of print.
Bernard et al. (2009). An allelic mutant series of ATM3 reveals its key role in the biogenesis of cytosolic iron-sulfur proteins in Arabidopsis. Plant Physiol. 151:590-602.
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 (Abcam), 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.
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