EF1A | elongation factor 1-alpha / EF-1-alpha protein
AS11 1633P | EF-1-alpha protein
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10 µg total protein extracted from 15 day old Arabidopsis thaliana seedlings that were either kept at 22°C (3) or heat treated at 38°C for 2 hours prior to protein extraction (4). As positive control 10 ng of recombinant AtEF1A (2) were separated side by side with the plant samples on 10-15 % gradient 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: 1000 for O/N 4°C 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 Agrisera, AS09 602, diluted to 1:35 000 for 1h at room temperature with agitation then washed as above and treated with ECL detection reagent according to the manufacturer's instructions. Exposure time was 5 seconds.
Lane (1) with no signal: recombinant Triticum spp. EF1A.
Courtesy Dr. Eman Basha, Arizona State University
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