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> Anti-V-ATPase - Epsilon subunit of tonoplast H+ATPase (affinty purified, goat antibody)

Anti-V-ATPase - Epsilon subunit of tonoplast H+ATPase (affinty purified, goat antibody)

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Katalog-Nummer AS09577A

Size : 200ug

Marke : Agrisera

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Anti-V-ATPase | Epsilon subunit of tonoplast H+ATPase (affinty purified, goat antibody)

Product no: AS09 577A

AS09 577A |  Clonality: Polyclonal  |  Host: Goat  |  Reactivity: Higher plants including A.thaliana, A.strigosa, M. truncatula, N. tabacum, S. lycopersicum

    Product Info
    Immunogen:

    KLH-conjugated synthetic peptide chosen from subunit E of plant V-ATPase including Arabidopsis thaliana At4g11150. Peptide is conserved in vacuolar H+-ATPase subunit E, isoform 1 to 3 (VHA-E1).
    Host: Goat
    Clonality: Polyclonal
    Purity: Immunogen affinity purified serum in PBS pH 7.4.
    Format: Lyophilized
    Quantity: 200 µg
    Reconstitution: For reconstitution add 100 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 the tubes briefly prior to opening them to avoid any losses that might occur from material adhering to the cap or sides of the tube.
    Tested applications: Immunofluorescence (IF), Western blot (WB)
    Recommended dilution: 1: 600 (IF), 1 : 1000-1 : 3000 (WB)
    Expected | apparent MW:

    26 | 31 kDa (Arabidopsis thaliana)
    Reactivity
    Confirmed reactivity: Arabidopsis thaliana, Avena strigosa, Medicago truncatula, Nicotiana tabacum, Oryza sativa, Solanum lycopersicum
    Predicted reactivity: Algae, Chlamydomonas reinhardtii, Hordeum vulgare, Malus domestica, Mesembryanthemum sp.,   Petunia sp.,Phaseolus sp. , Physcomitrium patens,  Pteris vittata (fern), Ricinus communis, Thellungiella sp., Zea mays, Vitis vinifera      Bull frog, Chicken, Bovine, Drosophila melanogaster, Human, Mouse, Rat
    Species of your interest not listed? Contact us
    Not reactive in: No confirmed exceptions from predicted reactivity are currently known
    Application Examples

    Immunofluorescent localization of V-ATPase epsilon subunit of tonoplast H+ATPase in suspension culture of Oryza sativa ssp. japonica cv. 'Unggi 9', using goat anti-V-ATPase, epsilon subunit of tonoplast antibodies (AS09 577A) and donkey anti-Goat IgG (H&L), DyLight® 488 conjugated secondary antibodies (AS10 1116, Agrisera).  Vacuolar membrane, tonoplast, is highlighted by yellow arrowheads. DAPI staining of nuclei is pseudocolored red. 
    Method
    Material: Suspension cultures of Oryza sativa ssp. japonica cv. 'Unggi 9
    Fixation: Packed cell volume to fixer ratio: 250 µl : 5ml
    Fixer composition and buffer: 4% (w/v) paraformaldehyde (freshly prepared as 8% stock and 0.2 µm filtered) 0.01% (v/v) Triton-X100 in Phosphate Buffered Saline (PBS), pH 7.4 (2x stock, 0.2 µm filtered)
    Container and method: in 6 cm Petri dish, gentle shaking at room temperature (RT)
    Duration: 40 min
    Hydrophilization: No
    Cell wall digestion: Yes Packed cell volume to enzyme ratio: 100 µl : 2 ml Enzyme composition: 1% (A) 1.2% (R) Cellulase (chromatically purified, powder, Worthington) 1% (A) 1.2% (R) Pectinase (protease free, liquid, Sigma) Buffer: 0.5% (w/v) MES buffer, pH 5.6
    Container and method: in 2 ml microfuge tube by rolling at room temperature (RT)
    Duration: 60 min
    Membrane permeabilization: Triton-X100 (0.35%), 7 min/RT
    Antigen retrieval: No
    Blocking buffer: Fish gelatin (5% v/v)
    Washing buffer: PBS
    Primary antibody dilution and incubation time: 1:600, 4ºC/ON
    Secondary antibody: donkey anti-Goat IgG (H&L), DyLight® 488 conjugated secondary antibodies (AS10 1116, Agrisera), 1:600, 1h/RT
    Co-staining of the nucleus (DAPI): Yes
    Cell wall and nucleus staining: 100 ng/ml DAPI

    Courtesy of Dr. Ferhan Ayaydin, Hungarian Centre of Excellence for Molecular Medicine (HCEMM), Szeged, Hungary
    Additional Information
    Additional information (application):

    V-ATPase is very sensitive for the redox of the SDS buffer. We recommend using at least 50-100 mM DTT freshly prepared before handling the sample.

    2 hours incubation with primary antibody is recommended over over night incubation which can contribute to increased background.
    Background
    Background:

    Plant vacuole V-ATPase is responsible for energization of transport of ions and metabolites, and acts as well 'house-keeping' and as a stress response enzyme. V-ATPase is a multi-subunit enzyme composed of a membrane sector and a cytosolic catalytic sector. It is related to the FoF1 ATP synthase. Alternative protein names: Vacuolar proton pump subunit E, Protein EMBRYO DEFECTIVE 2448
    Protocols Agrisera Western Blot protocol and video tutorials

    Protocols to work with plant and algal protein extracts


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