AOX1/2 | Plant alternative oxidase 1 and 2

PRODUCT INFORMATION IN PDF

product information Background 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. Immunogen 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 Host Rabbit Clonality Polyclonal Clone Purity Serum Format Lyophilized Quantity 50 µl Reconstitution For reconstitution add 50 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 Immunolocalization (IL), Western blot (WB) Related products AS06 152  AOX1 | alternative oxidase from Chlamydomonas reinhardtii, rabbit antibody AS10 699 AOX | alternative oxidase monoclonal antibody AS04 054PRE | AOX1/2 | plant alternative oxidase 1 and 2, pre-immune serum AS04 054S | AOX | AOX positive control/quantitation standard Recommended secondary antibody (for western blot with ECL detection) Plant protein extraction buffer Secondary antibodies Additional information Mitochondrion inner membrane marker. Possibly in the inner surgace of the inner mitochondrial membrane. Protocol for a plant mitochondria preparation can be found here.

application information
Recommended dilution		1 : 750 (IL), 1 : 1000 for 10-20 µg of mitochondrial protein/lane detection (WB)
Expected | apparent MW		36-40 | 36-40 for Arabidopsis thaliana
Confirmed reactivity		Arabidopsis thaliana, Betula nana, Beta vulgaris, Brassica napus, Kandelia candel, Eriphorum vaginatum, Hordeum vulgare, Lupinus luteus, Nicotiana tabacum, Picea abies, Pisum sativum, Poa annua, Robinia pseudoacacia, Solanum tuberosum, Solanum esculentum, Physcomitrella patens
Predicted reactivity		Aegilops tauschii, Brachypodium distachyon, Capsella rubella, Citrus sinensis, Citrus clementina, Corylus heterophylla, Crocus sativus, Cucumis sativus, Daucus carota, Glycine max, Hypericum perforatum, Lotus japonicus, Malus x domestica, Medicago truncatula, Medicago sativa, Nicotiana benthamiana, Oryza brachyantha, Oryza sativa, Populus tremula, Picea sitchensis, Triticum aestivum, Saccharum officinarum, Sauromatum venosum, Sorghum bicolor, Selaginella moellendorffii, Tetrahymena thermophila, Zea mays, Vigna unguiculata, Vitis vinifera
Not reactive in		Chlamydomonas reinhardtii
Additional information		According to Konert et al. (2015) AOX antibody is recognizing AOX1A and AOX1D. This product can be sold containing ProClin if requested.
Selected references		Vishwakarma et al. (2016). A discrete role for alternative oxidase under hypoxia to increase nitric oxide and drive energy production. Free Radic Biol Med. 2018 Mar 28. pii: S0891-5849(18)30148-5. doi: 10.1016/j.freeradbiomed.2018.03.045. Garmash et al. (2017). Expression profiles of genes for mitochondrial respiratory energy-dissipating systems and antioxidant enzymes in wheat leaves during de-etiolation. J Plant Physiol. 2017 Aug;215:110-121. doi: 10.1016/j.jplph.2017.05.023. Zhao et al. (2016). Nitrogen deprivation induces cross-tolerance of Poa annua callus to salt stress. Biologia Plantarum 60 (3): 543–554. Solti et al. (2016). Does a voltage-sensitive outer envelope transport mechanism contributes to the chloroplast iron uptake? Planta. 2016 Dec;244(6):1303-1313. Epub 2016 Aug 19. Zhang et al. (2016). A High Temperature-Dependent Mitochondrial Lipase EXTRA GLUME1 Promotes Floral Phenotypic Robustness against Temperature Fluctuation in Rice (Oryza sativa L.). PLoS Genet. 2016 Jul 1;12(7):e1006152. doi: 10.1371/journal.pgen.1006152. eCollection 2016. Meng et al. (2016). Physiological and proteomic responses to salt stress in chloroplasts of diploid and tetraploid black locust (Robinia pseudoacacia L.). Sci Rep. 2016 Mar 15;6:23098. doi: 10.1038/srep23098 Pavlovič et al. (2016). Light-induced gradual activation of photosystem II in dark-grown Norway spruce seedlings. Biochim Biophys Acta. 2016 Feb 18. pii: S0005-2728(16)30028-7. doi: 10.1016/j.bbabio.2016.02.009. 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.

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

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 standard ECL 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

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Plant protein extraction buffer

Secondary antibodies