LEA6-1 | Late embryogenesis abundant protein LEA6-1

Different amounts (25, 50, 100 and 400 ng) of the purified recombinant AtLEA6-1 protein in Laemmli buffer 1X were denatured at 75°C for 5 min, loaded and separated on 15 % SDS-PAGE. Gel was blotted 1h to nitrocellulose membranes (AmershamTM ProtamTM Premium 0.45 μM NC) using a tank transfer system (BIO-RAD). Blots were blocked ON with TBS-T/NF-Milk (5%) at 8 °C with agitation. Blot was incubated in the primary antibody at a dilution of 1: 500 for 1h at RT with agitation in TBS-T/NF-Milk (5%). The antibody solution was decanted, and the blot was rinsed briefly twice with TBS-T and then washed 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from Agrisera AS09 602) diluted to 1:10 000 in TBS-T/NF-Milk (5%) for 1h at RT with agitation. The blot was washed as above and developed for 3 min with a mix 1:1 of Peroxidase and Luminol (Thermo). Finally, membranes were exposed to X-ray films (Radiographic Blue films, Kodak) for 30’’ and developed using Kodak reagents.

Courtesy of Dr. Alejandra A. Covarrubias,Universidad Nacional Autónoma de México, Mexico

LEA (Late embryogenesis abundant) proteins are very hydrophilic proteins, described over 25 years ago as accumulating during late stages of plant seed development. Found in vegetative plant tissues following exposure to environmental stress. Synonymes: Putative late embryogenesis abundant protein LEA.