Purification of GST-HA-S6K: Difference between revisions
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updated lipofectamine conditions |
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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 | ||
Revision as of 18:18, 22 August 2012
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 293A cells into 10-150 mm dishes in DMEM/FBS with no antibiotics.
- Transfect when cells are 90-95% confluent.
- 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.
- Wait 20 min, then add ~2 mL to each plate of cells.
- After ~4h refeed with normal media (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
- 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.