Plant/Algal cell biology / Compartment markers / Vacuole

product information

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

KLH-conjugated synthetic peptide chosen from subunit E of plant V-ATPase including Arabidopsis thaliana At4g11150. Peptide is conserved in valuolar H+-ATPase subunit E, isoform 1 to 3 (VHA-E1).

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.

western blot, (WB) immunohistochemistry (IHC)

AS09 577 | anti-V-ATPase | epsilon subunit of tonoplast H+ATPase goat antibodies

marker antibodies for plant cellular compartments

cellular [compartment marker] of tonoplast membrane

application information

1 : 2 000 - 1: 5000 with alkaline phosphatase (WB), 1:50 (IHC)

26 | 31 kDa (Arabidopsis thaliana)

Ananas comosus, Arabidopsis thaliana, Chlamydomonas reinhardtii, Hordeum vulgare, Lycopersicum esculentum, Lilium longiflorum,  Mesembryanthemum sp. Nicotiana tabacum, Petunia sp. ,Pteris vittata (fern),Thellungiella sp. , Zea mays

dicots including Phaseolus sp. , Ricinus communis, Vitis vinifera and monocots including: Oryza sativa, Zea mays, algae, Physcomitrella patens,

bull frog, chicken, bovine, Drosophila melanogaster,human, mouse, rat

no confirmed exceptions from predicted reactivity known in the moment

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.

Immunostaining protocol using V-ATPase antibodies can be found here.

Pertl et al. (2009).  The pollen organelle membrane proteome reveals highly spatial-temporal dynamics during germination and tube growth of lily pollen. j. Proteome Res. 11:5142.5152. Hinton et al. (2009).  Function of a Subunit Isoforms of the V-ATPase in pH Homeostasis and in Vitro Invasion of MDA-MB231 Human Breast Cancer Cells. J.Biol.Chem. 24:16400-16408.  Reuveni et al. (2001). Decrease in vacuolar pH during petunia flower opening is reflected in the activity of tonoplast H+-ATPase. J. of Plant Physiol. 158:991-998.

application example

10 µg of total leaf protein extracted with PEB (AS08 300) from (1) Arabidopsis thaliana, (3) Zea mays, and (4) Hordeum vulgare together with (2) cytosolic extract from Arabidopsis thaliana leafs were separated on 4-12% NuPage (Invitrogen) LDS-PAGE and blotted 80 min (30V) to nitrocellulose. Filter was blocked 1h with 2% low-fat milk powder in TBS-T (0.1% TWEEN 20) and probed with anti-V-ATPase (AS07 213, 1:2000, 1h) and secondary anti-rabbit (1:40000, 1h) antibody (HRP conjugated, Abcam) in TBS-T containing 2% low fat milk powder. Antibody incubations were followed by washings in TBS-T (15, +5, +5, +5 min). All steps were performed at RT with agitation. Signal was detected with SuperSignal West Pico (Thermo Scientific) using a GenoPlex Chemi CCD (accumulated signal 10 x 30s exposure, bin 2x2).

application example

Standard IF protocol for plant material has been used including slight fixation with formaldehyde followed by washing and incubation with primary and secondary antibodies conjugated to fluorescent dyes. Green dye visualization of anti-V-ATPase antibody (Alexa 488 Molecular Probes), red – anti-tubulin antibody.