915
edits
Changes
updated lipofectamine conditions
==Protocol==
===Transfection of Cells===
#Split 293A cells into 10 new -150 mm dishes in DMEM/FBS with '''no antibiotics'''.#Transefect with Transfect when cells are 90-95% confluent.#Transfer cells to serum **and P/S/G free** media#Combine 240 500 ug DNA with 1.5 10 mL OptiMEM, and 600 separately 700 uL Lipofectamine 2000 with 1.5 10 mL OptiMEM
#After 5 minutes, combine the DNA solution with the Lipofectamine solution.
#Wait 20 min, then add 3 ~2 mL to each plate of cells.#After ~4h refeed with normal media (containing can now contain antibiotics)and incubate overnight.
===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)
#Add 0.5 mL [[HNTG Buffer]] per plate and scrape