Purification of GST-HA-S6K: Difference between revisions
updated lipofectamine conditions |
mNo edit summary |
||
| Line 13: | Line 13: | ||
#Split 293A cells into 10-150 mm dishes in DMEM/FBS with '''no antibiotics'''. | #Split 293A cells into 10-150 mm dishes in DMEM/FBS with '''no antibiotics'''. | ||
#Transfect when cells are 90-95% confluent. | #Transfect when cells are 90-95% confluent. | ||
#Combine 500 ug DNA with 10 mL OptiMEM, and separately 700 uL Lipofectamine 2000 with 10 mL OptiMEM | #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. | ||
| Line 22: | Line 21: | ||
#Treat cells for 30min with 100 nM Wortmannin to suppress endogenous S6K phosphorylation | #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]] | #Add 0.5 mL [[HNTG Buffer]] to each plate and scrape | ||
#Collect scraped cells and incubate end over end in eppendorf tube for 30 min | #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 | #Centrifuge in eppendorf centrifuge for 10 min at 14 000 RPM | ||