AOX1/2 | Plant alternative oxidase 1 and 2
AS04 054 | Clonality: Polyclonal | Host: Rabbit | Reactivity: A. thaliana, B. nana, B. napus, B. oleracea, 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
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Candidia albicans, Chlamydomonas reinhardtii (use an antibody to algal AOX1, AS06 152)
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
Reactant: Arabidopsis thaliana (Thale cress)
Application: Western Blotting
Pudmed ID: 32457324
Journal: Sci Rep
Figure Number: 2C
Published Date: 2020-05-26
First Author: Sako, K., Futamura, Y., et al.
Impact Factor: 4.13Open Publication
Expression profile of genes up-regulated by both FSL0260 treatment and salinity stress. (a) Cellular component gene ontology of up-regulated genes by FSL0260 treatment. (b) Relative expression levels of AtNDB4 and AtAOX1a genes during salinity-stress treatment for 0 and 2?h with or without 20?ĩM FSL0260. Expression level of plants treated with DMSO was set as 1. 18S rRNA was used as an internal standard. Error bars represent the mean ą SE (n?=?3). Statistical significance was determined by ANOVA, followed by post-hoc Tukey’s tests. Means that differed significantly (P?0.05) are indicated by different letters. (c) Immunoblot of the AOX (35?kDa) proteins (left). Coomassie blue-stained gel showing control loading (right). Total proteins were extracted from seedlings treated with 0 or 20?ĩM FSL0260 for 24?h and with or without subsequent treatment of 100?mM NaCl for 6?h. DMSO was used as a negative control. Immunoblot analysis was performed using an anti-AOX1/2 antibody. (d) The signal intensity of AOX1/2. DMSO treatment was taken as 1. Error bars represent the mean ą SE (n?=?3). Statistical significance was determined by ANOVA, followed by post-hoc Tukey’s tests. Means that differed significantly (P?0.05) are indicated by different letters.
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.
Pascual et al (2021). ACONITASE 3 is part of the ANAC017 transcription factor-dependent mitochondrial dysfunction response, Plant Physiology, 2021;, kiab225, https://doi.org/10.1093/plphys/kiab225
Challabathula et al. (2021) Differential modulation of photosynthesis, ROS and antioxidant enzyme activities in stress-sensitive and -tolerant rice cultivars during salinity and drought upon restriction of COX and AOX pathways of mitochondrial oxidative electron transport, Journal of Plant Physiology,Volume 268,2022,153583, ISSN 0176-1617,https://doi.org/10.1016/j.jplph.2021.153583.
Oh et al. (2021) Alternative oxidase (AOX) 1a and 1d limit proline-induced oxidative stress and aid salinity recovery in Arabidopsis. Plant Physiol. 2021 Dec 17:kiab578. doi: 10.1093/plphys/kiab578. Epub ahead of print. PMID: 34919733.
Pavlovic & Kocab. (2021) Alternative oxidase (AOX) in the carnivorous pitcher plants of the genus Nepenthes: what is it good for? Ann Bot. 2021 Dec 18:mcab151. doi: 10.1093/aob/mcab151. Epub ahead of print. PMID: 34922341.
Garmash et al. (2020). Altered levels of AOX1a expression result in changes in metabolic pathways in Arabidopsis thaliana plants acclimated to low dose rates of ultraviolet B radiation. Plant Sci. 2020 Feb;291:110332. doi: 10.1016/j.plantsci.2019.110332.
Makino et al. (2020). Induction of Terminal Oxidases of Electron Transport Chain in Broccoli Heads Under Controlled Atmosphere Storage. Foods, 9 (4)
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Tward et al. (2019). Identification of the alternative oxidase gene and its expression in the copepod Tigriopus californicus. Comp Biochem Physiol B Biochem Mol Biol. 2019 Feb;228:41-50. doi: 10.1016/j.cbpb.2018.11.003.
Réthoré et al. (2019). Arabidopsis seedlings display a remarkable resilience under severe mineral starvation using their metabolic plasticity to remain self-sufficient for weeks. Plant J. 2019 Mar 22. doi: 10.1111/tpj.14325.
Luévano-Martínez et al. (2019). Mitochondrial alternative oxidase is determinant for growth and sporulation in the early diverging fungus Blastocladiella emersonii. Fungal Biology, Vol 123, Issue 1, 59-65.
Córdoba et al. (2019). Different Types of CA Domains Are Present in Complex I from Immature Seeds and Adult Plants in Arabidopsis thaliana. Plant Cell Physiol. 2019 Jan 22. doi: 10.1093/pcp/pcz011.
Czobor et al. (2019). Comparison of the response of alternative oxidase and uncoupling proteins to bacterial elicitor induced oxidative burst. PLoS One. 2019 Jan 10;14(1):e0210592. doi: 10.1371/journal.pone.0210592.
Kuang et al. (2019). Quantitative Proteome Analysis Reveals Changes in the Protein Landscape During Grape Berry Development With a Focus on Vacuolar Transport Proteins. Front Plant Sci. 2019 May 15;10:641. doi: 10.3389/fpls.2019.00641. eCollection 2019.
Hu et al. (2018). OsNDUFA9 encoding a mitochondrial complex I subunit is essential for embryo development and starch synthesis in rice. Plant Cell Rep. 2018 Dec;37(12):1667-1679. doi: 10.1007/s00299-018-2338-x.
Borovik and Grabelnych (2018). Mitochondrial alternative cyanide-resistant oxidase is involved in an increase of heat stress tolerance in spring wheat. J Plant Physiol. 2018 Dec;231:310-317. doi: 10.1016/j.jplph.2018.10.007.
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.
Hu et al. (2018). OsNDUFA9 encoding a mitochondrial complex I subunit is essential for embryo development and starch synthesis in rice.Plant Cell Rep. 2018 Aug 27. doi: 10.1007/s00299-018-2338-x.
Zhu et al. (2018). Mitochondrial alternative oxidase-dependent autophagy involved in ethylene-mediated drought tolerance in Solanum lycopersicum. Plant Biotechnol J. 2018 May 4. doi: 10.1111/pbi.12939.
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.
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.
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
Pavlovi? 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'? 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.
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