ABI5 | Abscisic acid insensitive 5 (peptide antibody)
AS12 1863 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana
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2, 5 and 10 ug of total protein (run on separate lanes) extracted from the Columbia ecotype (Arabidopsis thaliana) seeds using Acetone extraction was separated using the Bolt® Bis-Tris Plus Gel system on a 4-12% gradient SDS-PAGE gel, blotted using the turbo-blot system (BIO-RAD) to transfer onto a PVDF membrane (7min). SNAP-ID (Millipore) system was used for blocking and antibody labeling. Blocking occured for 30 minutes (no agitation, 0.05 % skim milk in dest. water). Primary antibody labeling was done for 10 minutes at 1:200 dilution. Followed by 3x10 ml washes (PBST). Then blotted with secondary antibody (anti-rabbit IgG HRP conjugated from Agrisera AS09 602) for 10 min (1:1000 dilution) followed by three washes.Blot was developed using ECL as per manufacturer’s instructions. Exposure time was 2 min.
Courtesy of Nay Chi, Pogson Lab, Australia
MG132 is recommened to be added to extraction buffer as ABI5 is degraded by proteasome as well as homogenization with thiourea and bead beater.
Protocol for protein extraction from seeds can be requested here.
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