AKINB2 | SNF1-related protein kinase regulatory subunit beta-2

application example

25 µg of empty vector (1), pCDNA 3.1 – HA- tagged AKIN-beta1 (2), pCDNA 3.1 – HA- tagged AKIN-beta2 (3), 50 µg of total protein from Arabidopsis thaliana leaves had been homogenized into 50 mM NaPO4 pH 7.5, 20 mM KCl, 0.5 M sucrose, 0.2 mM PMSF, 10 mM DTT and protease inhibitor cocktail (Sigma, P9599). Extracts were separared on 10%NuPage Bis-Tris Novex SDS PAGE (Invitrogen) gels followed by a transfer for 60 min to PVDF membrane. Filters were blocked for 1h with 5% low-fat milk powder in PBS-T (0.1% TWEEN 20) and probed with anti-AKINbeta-2  antibodies (2 µg/ml for 60 min at RT) followed by Protein G-HRP (Bio-Rad; 1:3 000 for 60 min at RT) in PBS-T. Antibody incubations were followed by washings in PBS-T (3 x 5 min). All washing steps were performed at RT with agitation. Signal was detected with ECL (Millipore) using CCD camera. Exposure time was 10 seconds.

Courtesy Dr. David Stapleton