LEA6 | Late embryogenesis abundant protein 6

Samples were denatured with SDS DTT sample buffer (12 mM Tris pH 6.8, 10 % glycerol, 0.4 % SDS, 80 mM DTT, 0.02 % bromophenol blue) for 30 min at room temperature, then were separated on 12 % SDS-PAGE and blotted 1h at 100 V to PVDF membrane (pore size of 0.2 µm), using wet transfer (10 mM CAPS pH 11, 20% MeOH). Blot was blocked with 5% milk in TBS 0.1 % Tween20 for 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1 000 overnight/4°C in TBS 0.1 % Tween 20 without agitation. The antibody solution was decanted, and the blot was rinsed briefly twice, then washed 2 times for 30 min in TBS-T at RT with agitation. Blot was incubated in Agrisera matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:25 000 in 25 mL of TBS 0.1 %Tween20 for 1h/RT with agitation. The blot was washed as above and developed for 5 min with Bio-Rad Clarity western ECL substrate. Exposure time was 60 seconds.

Note: The apparent molecular mass (32-34 kDa) is 4 times higher than expected (8 kDa). This is consistent with publications that reported the aberrant behavior of some LEA proteins in SDS-PAGE.

Courtesy of Dr. David Macherel, IRHS/Seedling Metabolism & Stress, France