eIF2-alpha | Eukaryotic translation initiation factor 2 subunit alpha
AS20 4371 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana
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1-4 14 days old Arabidopsis thaliana wild-type
5 - Arabidopsis thaliana gcn2 mutant
Arabidopsis thaliana whole seedlings were grinded in liquid nitrogen and 80 mg of tissue powder was resuspended in 100 µl of 0.5xSDS Laemmli buffer in a 1.5 ml tube. After 5 minutes of mixing at room temperature, tubes were centrifuged at 21 000 xg for 10 minutes at RT. For immunoblog analysis 10 µl of supernatant was separated on a 12.5 % (w/v) SDS-PAGE gel and electroblotted onto PVDF membrane. After 1 h of blocking at RT with 1x blocking buffer (TBS-T, 5 % non-fat milk and 0.2 % BSA), the membrane was washed with 1xTBS-T four times for 10 minutes each. Post washing, membrane was incubated with anti-eIF2alpha antibodies at 1: 5000 for 48 h at 4°C. Following the washing with TBS-T, 10 minutes each for six repeats, the membrane was incubated in the goat anti-rabbit IgG, HRP conjugated secondary antibodies for 1h at RT followed by washing with TBS-T 10 minutes each for ten repeats and the blot was developed with chemiluminescent detection reagents, according to manufacture's recommendations.
Courtesy of Dr. Ansul Lokdarshi, Biochemistry & Cellular and Molecular Biology, University of Tennessee, USA
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