Plant/Algal cell antibodies / Populus sp.

product information

Alternative oxidases (AOX) are quinol oxidases located in the inner mitochondrial membrane of plants. They function as terminal oxidases in the alternate electron transport pathway, oxidizing ubiquinone to reduce oxygen to water.

KLH-conjugated synthetic peptide derived from fully conserved C-terminal consensus motif from plant AOX isoforms 1 and 2 including Arabidopsis thaliana AOX1A (At3g22370) and AOX2 (At5g64210), Solanum lycopersicum Q7XBG9, Oryza sativa Q7XT33,

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.

western blot (WB)

AS06 152  AOX1 | alternative oxidase from Chlamydomonas reinhardtii

AS10 699 AOX | alternative oxidase monoclonal antibody

Recommended secondary antibody (for western blot with ECL detection)

Protocol for plant mitochondria isolation can be found here.

application information

1 : 1000 on 10-20 µg of mitochondrial protein/lane with standard ECL detection (WB)

36-40 | 36-40 for Arabidopsis thaliana

Arabidopsis thaliana, Lupinus luteus, Pisum sativum, Solanum tuberosum, Physcomitrella patens

dicots including: Nicotiana tabacum and Glycine max, monocots including Oryza sativa, S.officinarum  and Triticum aestivum, conifers, diatoms

Chlamydomonas reinhardtii

to be added when available

Lang, E.G.E., S.J. Mueller, S.N.W. Hoernstein, J. Porankiewicz-Asplund, M. Vervliet-Scheebaum, R. Reski (2010). Simultaneous isolation of pure and intact chloroplasts and mitochondria from moss as basis for sub-cellular proteomics. Plant Cell Reports, DOI: 10.1007/s00299-010-0935-4. (open source)

Yamasaki et al. (2007) Regulation of Copper Homeostasis by Micro-RNA in Arabidopsis. J. Biol Chem 282: 16369-16378

application example

25 μg of Arabidopsis thaliana mitochondrial wild type fraction (1) mitochondrial fraction from a mutant with increased AOX level (2), total wild type leaf extract (3), total leaf extract from AOX overproducing mutant (4)  were separated on 10% gel and blotted on nitrocellulose membrane using wet transfer (0.22% CAPS, pH 11). Filters where blocked (1.5h) in 5% milk in TBST (1X TBS, 0,1% Tween 20), incubated with 1: 1000 anti-AOX polyclonal antibodies (2h in TBST) followed by 1 h incubation with 1: 50 000 Agrisera secondary anti-rabbit HRP-coupled antibodies (AS09 602) and visualized with standard ECL on Kodak autoradiography film for 15-60 s. Mitochondria were isolated as described by Urantowka et al. (Plant Mol Biol, 2005, 59:239-52). Mitochondrial pellets were suspended in 1X Laemmli buffer (5% beta-mercaptoetanol, 3.7% glycerol, 1.1% SDS, 23 mM Tris- HCl pH 6.8, 0.01% bromophenol blue), heated (95°C, 5 min.) and centrifuged (13 000rpm, 1 min.). Leaf extracts were prepared as described by Martinez-Garcia et al. (Plant J., 1999, 20:251-7).
Courtesy Dr. Janusz Piechota, Wrocław University, Poland