Delta-VPE | Vacuolar-processing enzyme delta-isozyme
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Arabidopsis thaliana maturing siliques was freshly extracted to a crude extract with 2x SDS-sample buffer (+ 2ME) for SDS-PAGE and denatured with 4X SDS buffer at 95°C for 5 min. were separated on 15-20 % 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: 5000 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.
Left panel: differential Interference Contrast Image
Right panel: Immunofluorescent staining
Material: developing seeds of Arabidopsis thaliana
Fixation: 7.2% (w/v) formaldehyde, 0.1% (v/v) Nonidet P-40, 10% (v/v) dimethyl sulfoxide, and 50 mM Na-phosphate buffer, pH 7.2 for 40 minutes.
Wash, 2x: TBS-T for 5 min, incubated in TBS-T containing 5% (w/v) Cellulase Onozuka R-10 and 2% (w/v) Pectolyase Y-23 for 20 min at 30°C
Was: 2x with TBS-T
Blocking: 30 min. in 2 % BSA in TBS-T
Primary antibody: 1: 500, 40 min.
Wash: 3x5 min. with TBS-T
Secondary antibodies: goat anti-rabbit AlexaFluor 488 conjugated
Wash: 3x 5 min. TBS-T and mounted
Kunieda et al. (2008). NAC family proteins NARS1/NAC2 and NARS2/NAM in the outer integument regulate embryogenesis in Arabidopsis. Plant Cell. 2008 Oct;20(10):2631-42.doi: 10.1105/tpc.108.060160. (Western blot, Arabidopsis thaliana)
Nakaune et al. (2005). A vacuolar processing enzyme, deltaVPE, is involved in seed coat formation at the early stage of seed development. Plant Cell. 2005 Mar;17(3):876-87. doi: 10.1105/tpc.104.026872. (Immunofluorescence, Immunolocalisation by electron microscopy, Western blot, Arabidopsis thaliana)
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