eIF2-alpha-P | Phosphorylated Eukaryotic translation initiation factor 2 subunit alpha

Samples:
Lane 1 (not shown, marker does not appear in chemiluninescence image): Bio-Rad Precision Plus All Blue prestained protein ladder
Lane 2: Arabidopsis thaliana Ler wt seedlings treated with glyphosate for an unknown time (positive control for eIF2alpha phosphorylation)
Lane 3: Arabidopsis thaliana Ler gcn2 -/- seedlings treated with glyphosate for an unknown time (negative control for eIF2alpha phosphorylation)
Lanes 4-9 are the C24 ecotype, with lane 4 being the control C24 seedlings and lanes 5-9 being stressed Lanes 10-15 are the Col-0 ecotype, with lane 10 being the control Col-0 seedlings and lanes 11-15 being stressed Lane
5-15: Arabidopsis thaliana C24 wt seedlings treated with various stresses

Five Arabidopsis thaliana seedlings (6 days old) per sample were collected & stored at -80 until use. Each sample was ground in liquid nitrogen, and total protein was extracted using 1x SDS-PAGE sample buffer. Samples were denatured at 70°C for 10 minutes and separated on a 10% gel using SDS-PAGE. Transfer to PVDF membrane was done at 10V for 110 minutes using semi-dry transfer. Blot was blocked with Bio-Rad EveryBlot blocking buffer for 2 hours at room temperature. Blot was incubated with primary antibody (AgriSera #AS20 4447) at 1:1000 in EveryBlot buffer at 4°C overnight, then washed for 3 x 20 minutes with 1x TBS-T with agitation. Blot was incubated with secondary antibody (AgriSera #AS09 602) at 1:10,000 in EveryBlot buffer at room temperature for 2 hours, then washed for 3 x 20 minutes with 1x TBS-T with agitation. Blot was developed for 5 minutes with AgriSera ECLBright and exposed for 2 minutes on the Bio-Rad ChemiDoc imaging system.

Courtesy of Dr, Anna Nelson Dittrich, Boyce Thompson Institute, USA