MTORC1 Kinase Assay: Difference between revisions
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*Protein A/G Beads | *Protein A/G Beads | ||
*[[CHAPS Lysis Buffer]] | *[[CHAPS Lysis Buffer]] | ||
*[[ | *[[TORC1 Kinase Buffer]] | ||
==Protocol== | ==Protocol== | ||
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*Add 15 uL Protein A/G Beads to tubes and incubate end over end for 1h at 4C | *Add 15 uL Protein A/G Beads to tubes and incubate end over end for 1h at 4C | ||
*Spin down beads for 15s at 14 000 RPM and aspirate with a fine-tipped (or crushed tip) pipet tip | *Spin down beads for 15s at 14 000 RPM and aspirate with a fine-tipped (or crushed tip) pipet tip | ||
*Wash beads 2x with Lysis Buffer and 1x with Kinase Buffer (-ATP) | *Wash beads 2x with Lysis Buffer and 1x with [[TORC1 Kinase Buffer]] (-ATP) | ||
*Add ATP (250 uM) and GST-S6K (200ng/rxn) to kinase buffer , 20 uL per reaction. | *Add ATP (250 uM) and GST-S6K (200ng/rxn) to kinase buffer , 20 uL per reaction. | ||
*Incubate at 30C for 30 min. | *Incubate at 30C for 30 min. | ||