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H+ATPase | Plasma membrane H+ATPase (rabbit antibody)

AS07 260 | Clonality: Polyclonal | Host: Rabbit | Reactivity: [global antibody] for di- and monocots, conifers, ferns, mosses, green algae | Cellular [compartment marker] for plasma membrane

H+ATPase | Plasma membrane H+ATPase (rabbit antibody) in the group Plant/Algal Antibodies / Arabidopsis thaliana  at Agrisera AB (Antibodies for research) (AS07 260)

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product information
Background

The Plasma Membrane H+ATPase is a family of proteins of ca. 100 kDa that are believed to be exclusive to the plasma membranes of plants and fungi. The protein is anchored within biological membrane which creates an electrochemical gradient used as an energy source and is essential for uptake of most metabolites and plant responses to environment, for example movement of leaves.

Immunogen

KLH-conjugated synthetic peptide exposed to cytoplasm in H+ATPase model, derived from available di and monocot, fern, mosses and algal plasma membrane ATPase sequences including Arabidopsis thaliana ATPase 1 (UniProt: P20649, TAIR: At2g18960) and ATPase 2 (UniProt: P19456 , TAIR: At4g30190), 3 (UniProt: P20431, TAIR: At5g57350), 4 (UniProt: Q9SU58, TAIR: At3g47950), 6 (UniProt: Q9SH76, TAIR: At2g07560), 7 (UniProt: Q9LY32, TAIR: At3g60330), 8 (UniProt: Q9M2A0, TAIR: At3g42640), 9 (UniProt: Q42556, TAIR: At1g80660), 11 (UniProt: Q9LV11, TAIR: At5g62670) of Arabidopsis thaliana and hydrogen ATPase of Chlamydomonas reinhardtii (Q9FNS3)

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. Do not Store this antibody in 4°C.
Tested applications Immunofluorescence (IF), Immunolocalization (IL), Western blot (WB)
Related products

AS07 213 | anti-V-ATPase rabbit antibody
AS07 260PRE | H+ATPase | plasma membrane H+ATPase, pre-immune serum
AS13 2671 | H+ATPase plasma membrane H+ATPase (chicken antibody)
Antibodies to membrane transport system
Recommended secondary antibody

Additional information

Cellular [compartment marker] for plasma membrane

application information
Recommended dilution 1 : 600-1 : 1000 (IF), 1 : 100 (IL), 1 : 1000-1 : 10 000 (WB)
Expected | apparent MW

90- 95 kDa (Arabidopsis thaliana, depending upon an isoform)

Confirmed reactivity Aesculus hippocastanum, Arabidopsis thaliana, Chlamydmonas reinhardtii, Cucumis sativus, Cucurbita moschata, Glycine max (weak), Kandelia obovata, Hordeum vulgare, Lolium perenne, Lycopersicon esculentum, Malus x domestica Borkh. c.v. Fuji, Marchantia polymorpha, Medicago truncatula, Nicotiana benthamiana, Nicotiana tabacum, Noccaea caerulescens, Oryza sativa, Petunia hybrida, Phalenopsis Sogo Yukidian cultivar V3, Physcomitrella patens, Picea abies, Populus tremula, Pteris vittata (fern), Ricinus communis, Spinacia oleracea, Zea mays, Vicia faba
Predicted reactivity

Algae, Avena sativa, Dunaliella spp., Gossypium hirsutum, Hordeum vulgare, Ostreococcus spp., Pinus thunbergii,Physocomitrella patens, Mesembruanthemum crystallinum, Mortierella elongata, Saccharomyces cerevisiae, Solanum tuberosum, Ulva prolifera

Not reactive in

Aspergillus niger

Additional information
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.

H+ATPase will be less abundant in mature roots and leafs. 

This product can be sold with ProClin if requested.
Selected references Seguel et al. (2018). PROHIBITIN 3 forms complexes with ISOCHORISMATE SYNTHASE 1 to regulate stress-induced salicylic acid biosynthesis in Arabidopsis. Plant Physiol. Jan 2018. DOI:10.1104/pp.17.00941
Duan et al. (2017). A Lipid-Anchored NAC Transcription Factor Is Translocated into the Nucleus and Activates Glyoxalase I Expression during Drought Stress. Plant Cell. 2017 Jul;29(7):1748-1772. doi: 10.1105/tpc.17.00044. (Nicotiana benthamiana)
Nagel et al. (2017). Arabidopsis SH3P2 is an ubiquitin-binding protein that functions together with ESCRT-I and the deubiquitylating enzyme AMSH3. Proc Natl Acad Sci U S A. 2017 Aug 7. pii: 201710866. doi: 10.1073/pnas.1710866114.
Aloui et al. (2017). The plasma membrane proteome of Medicago truncatula roots as modified by arbuscular mycorrhizal symbiosis. Mycorrhiza. 2017 Jul 19. doi: 10.1007/s00572-017-0789-5.
Lomin et al. (2017). Studies of cytokinin receptor–phosphotransmitter interaction provide evidences for the initiation of cytokinin signalling in the endoplasmic reticulum. Functional Plant Biology, CSIRO Publications. (Nicotiana benthamiana, western blot)
Kovaleva et al. (2017). Regulation of Petunia Pollen Tube Growth by Phytohormones: Identification of Their Potential Targets. DOI:10.17265/2161-6256/2016.04.004. (immunolocalization)
Liao et al. (2017). Arabidopsis E3 ubiquitin ligase PLANT U-BOX13 (PUB13) regulates chitin receptor LYSIN MOTIF RECEPTOR KINASE5 (LYK5) protein abundance. New Phytol. 2017 Feb 14. doi: 10.1111/nph.14472.
LaMontagne et al. (2016). Isolation of Microsomal Membrane Proteins from Arabidopsis thaliana. Curr. Protoc. Plant Biol. 1:217-234. doi: 10.1002/cppb.20020.

Application example

western blot using plant anti-H+ATPase antibodies

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.

Immunolocalization

immunolocalization using anti-H+ATPase polyclonal antibodies

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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