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Delta-VPE | Vacuolar-processing enzyme delta-isozyme

AS20 4408 | Clonality: Polyclonal  |  Host: Rabbit |  Reactivity: Arabidopsis thaliana
Delta-VPE | Vacuolar-processing enzyme delta-isozyme in the group Antibodies for Plant/Algal  / Membrane Transport System / Vacuolar membrane at Agrisera AB (Antibodies for research) (AS20 4408)

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Product Information

Immunogen Recombinant, His6-tagged, delta-VPE from Arabidopsis thaliana, UniProt: Q9LJX8, TAIR: At3g20210
Host Rabbit
Clonality Polyclonal
Purity Total IgG, purified on Protein A
Format Liquid at 2 mg/ml in PBS, 50% glycerol. Filter sterilized. No preservative or carrier protein.
Quantity 100 µg
Storage Store at -20°C; once make aliquots to avoid repeated freeze-thaw cycles. Please, remember to spin tubes briefly prior to opening them to avoid any losses that might occur from material adhering to the cap or sides of the tubes.
Tested applications Immunofluorescence (IF), Immunolocalisation (IL), Western blot (WB)
Recommended dilution 1: 500 (IF), 1: 5000 (IL), 1: 5000 (WB)
Expected | apparent MW 52 kDa (inactive precursor) | 37-38 kDa (mature, active form)

Reactivity

Confirmed reactivity Arabidopsis thaliana
Predicted reactivity Camelina sativa, Capsella rubella, Eutrema salsugineum, Raphanus sativus Species of your interest not listed? Contact us
Not reactive in No confirmed exceptions from predicted reactivity are currently known.

Application examples

Application examples Western blot using anti-Delta VPE antibodies

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.

Immunolocalisation using anti-delta VPE antibodies


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

Additional information

Subcellular localisation is seed specific and restricted to developing seeds at 7 days after anthesis. Delta-VPE is detected at lower levels in flowers siliques, specifically in seed coats.

Related products

Background

Background Delta VPE (Vacuplar-Processing Enzyme delta-isozyme) is Asparagine specific endopeptidase that may be involved in processing of proteins targeted to vacuoles (By similarity). The enzyme is probably involved in post-translational proteolysis of seed storage proteins in the protein storage vacuole of developing seeds (PubMed:12417707, PubMed:14688293). Exhibits a caspase-1-like activity in extracellular granules. At the early stage of seed development, required for the formation of the seed coat, by regulating cell death of specific cell layers in inner integument (Nakaune et al. 2005).

Product citations

Selected references Kunieda et al. (2013). Spatiotemporal secretion of PEROXIDASE36 is required for seed coat mucilage extrusion in Arabidopsis. Plant Cell . 2013 Apr;25(4):1355-67.doi: 10.1105/tpc.113.110072. (Western blot, Arabidopsis thaliana)
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)

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