AS20 4406 Anti-ALEU | Thiol protease aleurain

Arabidopsis thaliana crude extract was prepared from 7 day-old seedlings (1) 19 day-old seedlings (2) which were 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: 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.

The ALEU protein is synthesized as preproprotein with 39 kDa, the signal peptide of 21 amino acids is removed and the propeptide of N-terminal 119 amino acids are removed and not present in the mature, functional protein. The size of apparent molecular mass analyzed by Western blot is therefore 24 kDa.

Aleu protein may play a role in proteolysis leading to mobilization of nitrogen during senescence and starvation. Catalytic activity: Hydrolysis of proteins, acting as an aminopeptidase (notably, cleaving Arg-|-Xaa bonds) as well as an endopeptidase. Cellular localisation: vacuole. 

Alternative names: Senescence-associated gene product 2

1. Takagi et al. et al. (2013). MAIGO5 functions in protein export from Golgi-associated endoplasmic reticulum exit sites in Arabidopsis. Plant Cell. 2013 Nov;25(11):4658-75. doi: 10.1105/tpc.113.118158. (Western blot, Arabidopsis thaliana)
2. Ueda et al. (2006). AtVAM3 is required for normal specification of idioblasts, myrosin cells. Plant Cell Physiol. 2006 Jan;47(1):164-75. doi: 10.1093/pcp/pci232. (Western blot, Arabidopsis thaliana)

Agrisera Western Blot protocol and video tutorials

Protocols to work with plant and algal protein extracts

Agrisera Educational Posters Collection