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Gel-Filtration Chromatography

AL Alessio Lanna
DG Daniel C O Gomes
BM Bojana Muller-Durovic
TM Thomas McDonnell
DE David Escors
DG Derek W Gilroy
JL Jun Hee Lee
MK Michael Karin
AA Arne N Akbar
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A Superdex 200 (GE Healthcare) gel filtration column was first equilibrated with 1.5 CV of endotoxin free PBS (Hyclone) at room temperature. Sestrin2 complexes were injected at time 0 (500 ul) and eluted using a single isocratic wash, fractions collected, and analyzed by ELISA-based binding assays (absorbance at 450 nm) across the spectrum for the indicated proteins. Phosphorylated MAPKs were detected using Phospho-Tracer ELISA kits (Abcam) as above described. For detection of Mios (MBS9330396); RagA (MBS9334409); and RagB (MBS9318748). The column was calibrated with protein markers as described 18

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