VPS29 | Vacuolar protein sorting 29

Application example




50 µg of total protein from Arabidopsis thaliana suspension culture (1), 50 µg of unpurified total protein from Arabidopsis thaliana (2), 50 µg of total protein from Nicotiana tabacum leaf protoplasts (3), 20 µl of unpurified total leaf extract from Nicotiana tabacum (4), extracted with buffer containing 100 mM Tris (pH 7.8), 200 mM NaCl, 1 mM EDTA, 2% (v/v) beta-mercaptoethanol and 0.2% (v/v) Triton X-100, were separated on 10% SDS-PAGE and blotted 2h to nitrocellulose (semidry blot at 200 mA and RT). Blots were blocked with 5% (w/v) milk powder and 1% (w/v) BSA for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1:5 000 over night at 4°C with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG HRP conjugated) diluted to 1:10 000 in blocking solution for 45 min. at RT with agitation. The blot was washed as above and developed for 5 min with ECL according to the manufacturer's instructions. Exposure time was 20 seconds.

Courtesy of Simone Früholz, University of Tuebingen, Germany

VPS29 (vacuolar protein sorting 29) is a part of a retromer-like protein complex involved in endosome to lysosome protein transport. Synonymes:Vacuolar protein sorting-associated protein 29, Protein MAIGO 1.