AMY | Alpha-amylase
AS10 712 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Oryza sativa
|Recommended dilution||1 : 5000 (WB)|
|Expected | apparent MW||
43-48 kDa (depending upon the isoform)
|Confirmed reactivity||Oryza sativa, Panicum virgatum
Cereals, Kalanchoe laxiflora
|Not reactive in||
to be added when available, antibody released in December 2010.
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 standard ECL. 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
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