ALEU | Thiol protease aleurain
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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.
Alternative names: Senescence-associated gene product 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)