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

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

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.

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