AMY | Alpha-amylase

Cereals, Hordeum vulgare, Kalanchoe laxiflora

Pinus strobus

Application examples

25 µg of total protein from Oryza sativa seedlings (from 1 day to 5 days of imbibition on filter paper at 28°C) extracted with an SDS Extraction Buffer (60mM Tris-HCl pH 8.0, 2% SDS, 1,5% Sucrose) were separated on XT CRITERION 10%Bis-Tris (BioRad) SDS-PAGE and blotted 1h to PVDF. Blot was blocked immediately in milk in TBS-T for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a diluition of 1: 5,000 in milk in TBS-T for 3h at RT with agitation. The antibody solution was decanted and the blot was rinsed briefly once, then washed once for 15 min and 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (goat anti-rabbit IgG HRP conjugated, AS09 602) diluited 1:20 000 in milk in TBS-T for 50 min at RT and then washed as above and developed for 3 min with chemiluminescent detection. Images of the blot were obtained using BioSpectrum AC Imaging System (UVP). Exposure time was 30 min. The arrow indicates alpha-amylase (between 47kDa and 50KDa as expected).

Courtesy Valeria Banti and prof. Pierodomenico Perata, PlantLab, Scuola Superiore Sant'Anna

α-Amylases are hydrolytic enzymes responsible for the mobilization of the starch into metabolizable sugars. This process provides the energy for the growth of roots and shoots and is crucial during germination of cereal seeds.These enzymes are coded by a multigene family and even thought other amylolytic enzyme participate in the process of starch breakdown, the contribution of α-amylase is the prerequisite for the initiation of this process.