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

Application example

6 µg of total SDS-extracted protein from Avena strigosa roots (R) and leaves (L) , were separated on NuPage LDS-PAGE 4-12% gradient acrylamide gel (Invitrogen) and blotted 1h to nitrocellulose. Filters were blocked 1h with 5% low-fat milk powder in TBS and probed with anti-V-ATPase antibodies (AS09 577 , 1:2000, 1h) and secondary anti-goat (1:5000, 1 h) antibody in TBS containing 5% low fat milk powder. Antibody incubations were followed by washings in TBS-T (containing 0.05% Tween-20, 0.1% Triton X-100) . All steps were performed at RT with agitation. Blots were scanned with a Typhoon scanner.

Courtesy Dr. Sam Mugford (JIC), UK

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