H+ATPase | Plasma membrane H+ATPase
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1: 1000 - 1: 5000 with standard ECL OR 1: 10 000 with ECL-Advance, enhanced chemiluminescence (WB), 1: 600 - 1: 1000 (IF), 1: 100 (IL)
|Expected | apparent MW||
95 kDa (Arabidopsis thaliana)
dicots including: Arabidopsis thaliana, Cucumis sativus, Cucurbita moschata, Glycine max (weak), Kandelia obovata, Lolium perenne, Lycopersicon esculentum, Malus x domestica Borkh. c.v. Fuji, Marchantia polymorpha, Medicago truntata, Nicotiana tabacum, Noccaea caerulescens, Phalenopsis Sogo Yukidian cultivar V3, Ricinus communis, Spinacia oleracea, monocots inlcuding: Hordeum vulgare, Oryza sativa, Zea mays, trees: Picea abies, Populus tremula, Pteris vittata (fern), algae: Chlamydmonas reinhardtii
dicots including: Mesembruanthemum crystallinum, Solanum tuberosum; monocots (including Avena sativa, Hordeum vulgare, conifers (Pinus thunbergii), mosses (Physocomitrella patens), algae (Dunaliella spp., Ostreococcus spp.), Saccharomyces cerevisiae
|Not reactive in||
for additional Western blot detection image please refer to the article below.
VERY IMPORTANT: please, do not heat up your samples over 70°C as this might cause H+ATPase to precipitate and there will be no signal on your western blot.
|Selected references||Liu et al. (2016). iTRAQ-based quantitative proteomic analysis reveals the role of the tonoplast in fruit senescence. J Proteomics. 2016 Sep 2;146:80-9. doi: 10.1016/j.jprot.2016.06.031.
Wattelet-Boyer et al. (2016). Enrichment of hydroxylated C24- and C26-acyl- chain sphingolipids mediates PIN2 apical sorting at trans-Golgi network subdomains. Nat Commun. 2016 Sep 29;7:12788. doi: 10.1038/ncomms12788.
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). Three SAUR proteins SAUR76, SAUR77 and SAUR78 promote plant growth in Arabidopsis. Sci Rep. 2015 Jul 24;5:12477. doi: 10.1038/srep12477.
Kolb et al. (2015). FYVE1 is essential for vacuole biogenesis and intracellular trafficking in Arabidopsis thaliana. Plant Physiol. 2015 Feb 19. pii: pp.114.253377.
Hu et al. (2015). Re-examination of chlorophyllase function implies its involvement in defense against chewing herbivores. Plant Physiol. 2015 Jan 12. pii: pp.114.252023.
Komatsu et al. (2014). Phototropin Encoded by a Single-Copy Gene Mediates Chloroplast Photorelocation Movements in the Liverwort Marchantia polymorpha L. 1. Plant Physiol. 2014 Sep;166(1):411-27. doi: 10.1104/pp.114.245100. Epub 2014 Aug 5.
Berthier et al. (2014).Identification of a new sucrose transporter in rye-grass (LpSUT2): Effect of defoliation and putative fructose sensing. Plant Physiol & Biochem, vol: 84, Nov. 2014. (immunolocalization, leaf sheath)
Migocka et al. (2014). Molecular and biochemical properties of two P 1B2 -ATPases, CsHMA3 and CsHMA4, from cucumber. Plant Cell Environ. 2014 Sep 11. doi: 10.1111/pce.12447.
Shen et al. (2014). The fronds tonoplast quantitative proteomic analysis in arsenic hyperaccumulator Pteris vittata L. J Proteomics. 2014 Feb 4. pii: S1874-3919(14)00047-5. doi: 10.1016/j.jprot.2014.01.029.
Zhang et al. (2014). Arabidopsis ABCG14 protein controls the acropetal translocation of root-synthesized cytokinins. Nat Commun. 2014 Feb 11;5:3274. doi: 10.1038/ncomms4274.
Sobrino-Plata et al. (2014). Glutathione is a key antioxidant metabolite to cope with mercury and cadmium stress. Plant Soil, DOI 10.1007/s11104-013-2006-4.
20 µg of total protein from Arabidopsis thaliana (1), Hordeum vulgare (2), Zea mays (3), Nicotiana tabaccum plasma membrane fraction, 2.5 µg (4), extracted with Protein Extration Buffer, PEB (AS08 300), were boiled for 10 min. in 70°C and separated on 4-12% NuPage (Invitrogen) LDS-PAGE and blotted 1h to PVDF. Blots were blocked immediately following transfer in 2% ECL Advance blocking reagent (GE Healthcare) in 20 mM Tris, 137 mM sodium chloride pH 7.6 with 0.1% (v/v) Tween-20 (TBS-T) for 1h at room temperature with agitation. Blots were incubated in the primary antibody at a dilution of 1: 5 000 for 1h at room temperature with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at room temperature with agitation. Blots were incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, recommended secondary antibody AS09 602) diluted to 1:20 000 in 2% ECL Advance blocking solution for 1h at room temperature with agitation. The blots were washed as above and developed for 5 min with ECL Advance detection reagent according the manufacturers instructions. Images of the blots were obtained using a CCD imager (FluorSMax, Bio-Rad) and Quantity One software (Bio-Rad). Exposure time was 2 min.
Plasma membrane H+ATPase localization inArabidopsis thaliana roots.
Arabidopsis thaliana, elongation zone, H+ATPase (green). Arabidopsis thaliana roots were fixed in para-formaldehyde for 30 minutes. Tissue cleaning has been performed before immunolocalization. Anti-rabbit H+ATPase | plasma membrane primary antibody diluted in 1: 300 and anti-rabbit IgG secondary antibody conjugated with Alexa 555. Co-staining with DAPI visualized nucleus (blue color). Scale bar – 100 µm.
Courtesy Dr. Taras Pasternak, Freiburg University
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