application example

10 µg total protein extracted from 15 day old Arabidopsis thaliana seedlings that were either kept at 22ºC (1) or heat treated at 38ºC for 2 hours prior to protein extraction (2). As positive control 10 ng of recombinant elongation factor proteins gamma 1 (3) and gamma 2 (4) were separated side by side with the plant samples on 11% SDS-PAGE and blotted to nitrocellulose (Bio-rad). Blots were blocked following transfer with 5% low fat milk in low salt buffer for 1h at room temperature with agitation. Blots were incubated in the primary antibody at a dilution of 1: 2000 for 2h at room temperature with agitation in the blocking solution. The primary antibody solution was removed and the blot was rinsed briefly twice, then washed 4 times for 15 min each at room temperature with agitation using low salt buffer. Blots were incubated in secondary antibody (anti-rabbit IgG horse radish peroxides conjugated, from Amersham diluted to 1:2500 for 1h at room temperature with agitation then washed as above and treated with ECL detection reagent according to the manufacturers instructions. Exposure time was 5 seconds.

Low salt buffer components are 10 mM Tris (pH 7.6), 68 mM NaCl and 0.05 % Triton X-100.