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==Materials==
*GST-HA-S6K1 plasmid. Prepare by [[Cesium Chloride Preparation of DNA]]. Need 240 ug per 10 plates of cells
*293A Cells.
*Glutathione-Sepharose
*[[HNTG Buffer]]
*[[TORC1 Kinase Buffer]]
*Lipofectamine 2000
*OptiMEM
==Protocol==
===Transfection of Cells===
#Split cells into 10 new dishes in DMEM/FBS with '''no antibiotics'''.
#Transefect with 90-95% confluent
#Combine 240 ug DNA with 1.5 mL OptiMEM, and 600 uL Lipofectamine 2000 with 1.5 mL OptiMEM
#After 5 minutes, combine the DNA solution with the Lipofectamine solution.
#Wait 20 min, then add 3 mL to each plate of cells
#After ~4h refeed with normal media (containing antibiotics)
===Purification of GST-S6K1===
#Wash cells with D-PBS -/- twice (10 mL per plate)
#Add 0.5 mL [[HNTG Buffer]] per plate and scrape
#Collect scraped cells and incubate end over end in eppendorf tube for 30 min
#Centrifuge in eppendorf centrifuge for 10 min at 14 000 RPM
#Collect supernatant (save a lysate sample) into a 15 mL Falcon tube
#Add 0.5 mL Glutatione-sepharose beads and incubate end over end for 1-2h.
*GST-HA-S6K1 plasmid. Prepare by [[Cesium Chloride Preparation of DNA]]. Need 240 ug per 10 plates of cells
*293A Cells.
*Glutathione-Sepharose
*[[HNTG Buffer]]
*[[TORC1 Kinase Buffer]]
*Lipofectamine 2000
*OptiMEM
==Protocol==
===Transfection of Cells===
#Split cells into 10 new dishes in DMEM/FBS with '''no antibiotics'''.
#Transefect with 90-95% confluent
#Combine 240 ug DNA with 1.5 mL OptiMEM, and 600 uL Lipofectamine 2000 with 1.5 mL OptiMEM
#After 5 minutes, combine the DNA solution with the Lipofectamine solution.
#Wait 20 min, then add 3 mL to each plate of cells
#After ~4h refeed with normal media (containing antibiotics)
===Purification of GST-S6K1===
#Wash cells with D-PBS -/- twice (10 mL per plate)
#Add 0.5 mL [[HNTG Buffer]] per plate and scrape
#Collect scraped cells and incubate end over end in eppendorf tube for 30 min
#Centrifuge in eppendorf centrifuge for 10 min at 14 000 RPM
#Collect supernatant (save a lysate sample) into a 15 mL Falcon tube
#Add 0.5 mL Glutatione-sepharose beads and incubate end over end for 1-2h.
