VDAC1-5 | Voltage-dependent anion-selective channel protein 1-5
AS07 212 | Clonality: Polyclonal | Host: Rabbit | Reactivity: A.thaliana, di and monocots | Cellular [compartment marker] of mitochondrial outer membrane
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KLH-conjugated peptide conserved in all known higher plant VDAC proteins including Arabidopsis thaliana VDAC1 UniProt: Q9SRH5 , TAIR: AT3G01280, VDAC2 UniProt F4K3R8-1 , TAIR: AT5G67500, VDAC3 UniProt: Q9SMX3-1, TAIR: AT5G15090, VDAC4 UniProt: Q9FKM2-1, TAIR:AT5G57490, VDAC5 UniProt: Q9M2W6-1, TAIR: AT3G49920
29 kDa (for Arabidopsis thaliana)
Arabidopsis alpina, Aundo donax, Brachypodium distachyon, Brassica campestris, Brassica napus, Brassica rapa subsp. pekinensis, Capsella rubella, Citrus clementina, Eutrema salsugineum, Glycine max, Glycine soja, Gossypium arboreum, Hoedum vulgare var. distichum, Jatropha curcas, Medicago truncatula, Mesembryanthemum crystallinum, Morus notabilis, Nicotiana tabacum, Phaseolus coccineus, Phaseolus vulgaris, Pisum sativum, Plantago major, Prunus persica, Ricinus communis, Solanum lycopersicum, Solanum tuberosum, Sorghum bicolor, Theobroma cacao, Triticum aestivum, Vitis vinifera, Zea mays
Chlamydomonas reinhardtii, Glycine max, Zea mays, diatoms, Saccharomyces cerevisiae
Crude membrane proteins were separated on 12% SDS-PAGE and blotted 1h to PVDF. Blots were blocked immediately following transfer in 5% blocking reagent (BioRad, 170-6404) in 50 mM Tris, 150 mM sodium chloride pH 7.5 with 0.1% (v/v) Tween-20 (TBS-T) for 1h at room temperature with agitation. Blots were incubated in the primary antibody in 1: 5000 dilution for over-night at 4°C with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed 3 times for 5 min in TBS-T at room temperature with agitation. Blots were incubated in secondary antibody (Goat anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:5000 in 0.2% blocking solution for 1h at room temperature with agitation. The blots were washed as above and developed for 1~2 min with chemiluminescent detection reagent, according the manufacturers instructions. Images of the blots were obtained using a CCD imager (LAS4000 GE) and by ImageQuant software (GE).
Fixation and Immunolocalization
(A) full confocal stacks; (B) Single confocal section
Pollen tubes were fixed in 400 μM 3-maleimodobenzoic acid N-hydroxysuccinimide ester (MBS, Pierce) for 6 min at 20ºC, followed by 2% formaldehyde (1 h, 4°C). Cells were washed three times in 1x TBS then once in MES buffer (15 mM MES, pH 5.0), then incubated in 0.05% cellulose/0.05% macerozyme with 0.1% Triton X-100 in MES buffer containing 0.1 mM PMSF and 1 % BSA for 15 min. Cells were washed once in MES, then twice in TBS and then incubated in blocking solution (1% BSA in TBS) for 30 min at room temperature. Pollen was incubated with anti-VDAC1 antibodyies diluted in blocking solution (at 1:500) overnight at 4°C. Following TBS washes pollen was then incubated with the secondary antibody for 1.5 h at room temperature followed by further TBS washes. Pollen tubes were mounted on slides with 5 μL of Vectashield + DAPI (Vector Laboratories, USA) and coverslips sealed with nail varnish.
Method taken from Poulter et al (submitted) Actin-binding proteins implicated in formation of the punctate actin foci stimulated by the self-incompatibility response in Papaver . Submitted to Plant Physiology.
Courtesy Professor Noni Franklin-Tong, University of Birmingham, UK
Cellular [compartment marker] of mitochondrial outer membrane for western blot.
VDAC proteins are porin-type, beta-barrel diffusion pores. Prominently localized in the outer mitochondrial membrane and involved in metabolite exchange.
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de Michele et al. (2016). Free-Flow Electrophoresis of Plasma Membrane Vesicles Enriched by Two-Phase Partitioning Enhances the Quality of the Proteome from Arabidopsis Seedlings. J Proteome Res. 2016 Mar 4;15(3):900-13. doi: 10.1021/acs.jproteome.5b00876. Epub 2016 Feb 4.
Li et al. (2015). A Chaperone Function of NO CATALASE ACTIVITY1 Is Required to Maintain Catalase Activity and for Multiple Stress Responses in Arabidopsis. Plant Cell. 2015 Feb 19. pii: tpc.114.135095.
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.
Hsueh et al. (2014). The chloroplast outer envelope protein P39 in Arabidopsis thaliana belongs to the Omp85 protein family. Proteins. 2014 Nov 17. doi: 10.1002/prot.24725.
Takahashi et al. (2014). Transport of rice cyclobutane pyrimidine dimer (CPD) photolyase into mitochondria relies on a targeting sequence located in its C-terminal internal region.
Alcántar-Aguirre et al.(2013).ATP produced by oxidative phosphorylation is channeled toward hexokinase bound to mitochondrial porin (VDAC) in beetroots (Beta vulgaris). Planta, March 17.