V-ATPase, a1 | vacuolar H+-ATPase subunit a isoform 1
AS14 2822 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana
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93 kDa (Arabidopsis thaliana)
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10 μg of microsomal membranes were isolated from 6-days-old etiolated (dark-grown) Arabidopsis thaliana seedlings with Extraction buffer (450mM sucrose, 50mM HEPES pH7.5, 5mM MgCl2, 1mM DTT, 1x protease inhibitor). Proteins were denaturated in SDS sample buffer for 5 min at 95°C. Proteins were separated on a 10% SDS-PAGE and blotted 1h to a nitrocellulose membrane using tank transfer. Blots were blocked with 4% dry milk in TBS-T for 2h at room temperature (RT) with agitation. Blot was incubated in the primary antibody (#AS14 2822) at a dilution of 1:1000 in blocking buffer for 16h with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed 3 times for 15 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from Agrisera #AS09 602) diluted to 1:25 000 in blocking buffer for 2h at RT with agitation. The blot was washed as above and developed for 3 min with ECL according to the manufacturer's instructions peqlab AceGlow Kit & INTAS digital imager. Exposure time was 3 min.
Courtesy of Fabian Fink, University Heidelberg, Germany
Protein or membrane sample should be treated at 70°C for 10 min before loading on the gel.
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