VSP | Vegetative storage protein 1
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Arabidopsis thaliana maturing siliques were freshly extracted with 100 mm Tris-HCl, pH 6.8, 2% [w/v] SDS, 40% [v/v] glycerol, and 2% [v/v] 2-mercaptoethanol for SDS-PAGE and denatured at 95°C for 5 min. Sample was separated on 15 % SDS-PAGE and blotted at 15V overnight using wet transfer to PVDF membrane. Blot was thoroughly dried before blocking 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 recommendation.
Blot below has been conducted with the same protocol, but ER bodies were induced in rosette leaves by treatment with MeJA.
Water treatment (1,2), treatment with 50 µM MeJA treatment (3,4); 50 µM MeJA plus 20 µl/L ethylene for 36h (5,6). Described in Matsushima et al. (2002).
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