Purified eIF2 was phosphorylated in vitro by human PKR (Invitrogen)35 (link). eIF2-eIF2B complexes were formed by adding increasing amounts of phosphorylated eIF2 to eIF2B immobilized on anti-FLAG and and prewashed with phosphorylation buffer (20 mM Tris (pH 7.5), 100 mM KCl, 10 mM MgCl2, 5 mM β-glycerophosphate, 2 mM dithiothreitol, 10% glycerine, 0.1% NP-40, 200 μM ATP). The mixture was incubated constantly rotating at RT for 30 min. The eIF2-eIF2B complex was washed with 500 mM ammonium acetate and eluted with FLAG peptide. Eluted fractions were additionally washed with 500 mM ammonium acetate and concentrated in an Amicon Ultra (100 K MWCO). Low salt (100 mM KCl) purification of FLAG-tagged eIF2B resulted in maintaining interactions with eIF2 and eIF2B:eIF2 complex was eluted from anti-FLAG resin19 (link).
Human pkr
Manufactured by Thermo Fisher Scientific
Human PKR is a laboratory equipment product. It is a protein kinase that plays a key role in the innate immune response to viral infection.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using human pkr
1
Phosphorylated eIF2-eIF2B Complex Formation
2
Eukaryotic Translation Initiation Factors Purification
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