Anti-ABA2 | ABA deficient 2

5-10 µg/well of frozen total protein extracted from Arabidopsis thaliana leaves (Col-0 & aba2-2). Exact buffer components were: (50 mM Tris-HCl pH 8.0, 10 mM NaCl, 1% SDS, 0.1 mM DTT, 0.5% 2-mercaptoethanol) and denatured with (50 mM Tris-HCl pH 6.8, 6% glycerol, 1.5% w/v DTT, bromophenol blue) at 99 °C/5 min. Samples were separated in the cold on 10 % SDS-PAGE and blotted for 30 min at 20V to PVDF (pore size of 0.2 um), using semi-dry in the cold. Blot was blocked with 5 % milk for: 1h/RT or with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1000 ON/4°C in TBS-T + 5% skim milk with agitation. The antibody solution was decanted, and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 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: 25 000 in TBS-T + 5% skim milk for 1h/RT with agitation. The blot was washed as above and developed with a following chemiluminescent detection reagent. Exposure time was 5 minutes.

Courtesy of Ben Brookbank from laboratory of prof. Eiji Nambara, University of Toronto, Canada