VPS35 | Vacuolar protein sorting-associated protein 35B (marker of PVC)
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Arabidopsis thaliana 19 day-old seedlings were extracted to a crude extract and separated on 12.5 % SDS-PAGE and blotted to PVDF membrane in wet system. Blot was blocked with 3 % skim milk/TBS-T, 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 2000 in TBS-T for 1h/RT. The antibody solution was decanted and the blot was washed 4 times for 10 min in TBS-T at RT with agitation. Blot was incubated in matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:10 000 in for 1h/RT with agitation. The blot was washed as above and developed with a chemiluminescent detection reagent, following manufacture's recommendations.
Immunofluorescent localisation of VPS35.
Tobacco NY-2 cells were transnformed with Arabidopsis thaliana VPS35 (left panel), PEP12 (middle panel, which is a PVC marker)
Method described in details in: Yamazaki et al. (2008).
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