Purification of GST-HA-S6K: Difference between revisions
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==Protocol== | ==Protocol== | ||
===Transfection of Cells=== | ===Transfection of Cells=== | ||
#Split cells into 10 | #Split 293A cells into 10-150 mm dishes in DMEM/FBS with '''no antibiotics'''. | ||
# | #Transfect when cells are 90-95% confluent. | ||
#Combine | #Transfer cells to serum **and P/S/G free** media | ||
#Combine 500 ug DNA with 10 mL OptiMEM, and separately 700 uL Lipofectamine 2000 with 10 mL OptiMEM | |||
#After 5 minutes, combine the DNA solution with the Lipofectamine solution. | #After 5 minutes, combine the DNA solution with the Lipofectamine solution. | ||
#Wait 20 min, then add | #Wait 20 min, then add ~2 mL to each plate of cells. | ||
#After ~4h refeed with normal media ( | #After ~4h refeed with normal media (can now contain antibiotics) and incubate overnight. | ||
===Purification of GST-S6K1=== | ===Purification of GST-S6K1=== | ||
#Treat cells for 30min with 100 nM Wortmannin to suppress endogenous S6K phosphorylation | |||
#Wash cells with D-PBS -/- twice (10 mL per plate) | #Wash cells with D-PBS -/- twice (10 mL per plate) | ||
#Add 0.5 mL [[HNTG Buffer]] per plate and scrape | #Add 0.5 mL [[HNTG Buffer]] per plate and scrape | ||