eEF1B-beta1 and 2 | elongation factor 1-beta1 and 1-beta2

PRODUCT INFORMATION IN PDF

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 beta 1 (3) and beta 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. The primary antibody could be reused if it is kept at 4ºC for 2 weeks and if frozen at -20ºC for long time.

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

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Plant protein extraction buffer

Secondary antibodies