AOX1/2 | Plant alternative oxidase 1 and 2
AS04 054 | Clonality: Polyclonal | Host: Rabbit | Reactivity: A. thaliana, B. nana, B. napus, B. vulgaris, E. vaginatum, H. vulgare, L. luteus, N.tabacum, O. sativa, P.abies, P. sativum, S.tuberosum and T. aestivum, conifers and P. patens cellular[compartment marker] of mitochondrial inner membrane
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KLH-conjugated synthetic peptide derived from fully conserved C-terminal consensus motif from plant AOX isoforms including Arabidopsis thaliana AOX1A UniProt: Q39219, TAIR: At3g22370, AOX1B UniProt: O23913, TAIR:AT3G22360, AOX1C UniProt: O22048, TAIR: AT3G27620, and AOX2, UniProt: O22049, TAIR: AT5G64210, Solanum lycopersicum UniProt: Q7XBG9, Oryza sativa UniProt: Q7XT33, AOX1D, TAIR: AT1G32350
36-40 | 36-40 for Arabidopsis thaliana
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 chemiluminescent detection reagent, 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
20 μg of mitochondrial protein isolated from 2-week-old Arabidopsis thaliana seedlings (Smakowska et al., 2016) extracted with a buffer containing urea, thiourea, CHAPS and Triton X-100 (Heidorn-Czarna et al., 2018) were denaturated with Laemmli buffer at 95°C for 5 min and separated on 12% SDS-PAGE. Wild-type grown at 22°C (1), mutant grown at 22°C (2), wild-type grown at 30°C (3), mutant grown at 30°C.
Afterwards the gel was blotted for 1.5h to nitrocellulose membrane using wet-transfer. Blot was blocked with 5% milk in TBS-T at 4°C/ON with agitation. Blot was incubated in the primary antibody (anti-AOX1/2, AS04 054) at a dilution 1:1000 in 5% milk in TBS-T for 1.5h /RT with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 2 times for min in TBS-T at RT with agitation.
Blot was incubated in Agrisera matching secondary antibody (goat anti-rabbit IgG, HRP-conjugated, AS09 602) diluted to 1:20 000 in 5% milk in TBS-T for 1h/RT with agitation. The blot was washed as above and developed with chemiluminescence using GBox imager (Syngene).
Courtesy Dr. Małgorzata Heidorn-Czarna, University of Wrocław, Poland
Lines C0, C1- 10 µg of cauliflower mitochondrial proteins (C0- controls; C1- plants grown in mild drought conditions) isolated as described by Rurek et al., 2015 (doi:10.1016/j.bbabio.2015.01.005) were separated by 12% SDS- PAGE and electroblotted in semi-dry conditions (Towbin buffer) to Immobilon-P membrane (Millipore). Blots were CBB R 250 briefly stained, destained, wet-scanned and after completed destaining, they were blocked in 5% skimmed milk (dissolved in PBS-T containing 0.1% Tween 20) in 1h, RT. Primary antisera (at 1: 1000, diluted in 2% skimmed milk in PBS-T) were bound by overnight incubation of blots at +4 O C. After blot washing (2 times quick, 2 times of 5 min, and 10 min at the end), secondary goat anti-rabbit IgGs, HRP- conjugated (Agrisera, AS09 602; at 1: 50 000, diluted in 2% milk/ PBS-T) were bound in 1 h, RT. Blots were washed (as above) with copious amounts of PBS-T and chemiluminescence signals acquired by using chemiluminescent detection reagents on RTG film between 3 s and 2 min (periods of the given image acquisition were indicated).
100 µg of cauliflower mitochondria were pelleted and proteins were digitonin solubilised (30 min at 4°C) at the detergent: protein ratio 4:1 (g:g) using ACA 750 buffer. Unsolubilised material was further pelleted and supernatant after complementation with Serva Blue was loaded onto 4.5-16% gradient BN gel. After separation, protein complexes in the gel were denatured and reduced (in the presence of SDS and 2-mercaptoethanol) and then they were electroblotted and immunodetected essentially in the same manner as it was indicated for SDS-PAGE blots. Four complexes containing alternative oxidase were detected (the most abundant ca.150 and 120 kDa). This data is very similar to the one obtained for green tissue mitochondria of Arabidopsis and Medicago (see Gelmap project; https://gelmap.de/). Mobility of known OHPHOS complexes (complex I, II, III, IV and ATP synthase= complex V) was additionally indicated.
Courtesy Dr. Michał Rurek, Department of Molecular and Cellular Biology, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University in Poznań, Poland
Mitochondrion inner membrane marker. Possibly in the inner surface of the inner mitochondrial membrane.
Protocol for a plant mitochondria preparation can be found here.
In protein samples which are older than few months AOX enzyme can undergo intensive dimerization. Such preparations should not be used to work with this antibody.
According to Konert et al. (2015) AOX antibody is recognizing AOX1A and AOX1D.
This product can be sold containing ProClin if requested.
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.
Umekawa and Ito (2018). Thioredoxin o-mediated reduction of mitochondrial alternative oxidase in the thermogenic skunk cabbage Symplocarpus renifolius. J Biochem. 2018 Oct 5. doi: 10.1093/jb/mvy082.
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Konert et al.(2015). Protein phosphatase 2A (PP2A) regulatory subunit B'γ interacts with cytoplasmic ACONITASE 3 and modulates the abundance of AOX1A and AOX1D in Arabidopsis thaliana. New Phytol. 2015 Feb;205(3):1250-63. doi: 10.1111/nph.13097. Epub 2014 Oct 13.