Purification of GST-HA-S6K

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

1. Split 293A cells into 10-150 mm dishes in DMEM/FBS with no antibiotics.
2. Transfect when cells are 90-95% confluent.
3. Transfer cells to serum **and P/S/G free** media
4. Combine 500 ug DNA with 10 mL OptiMEM, and separately 700 uL Lipofectamine 2000 with 10 mL OptiMEM
5. After 5 minutes, combine the DNA solution with the Lipofectamine solution.
6. Wait 20 min, then add ~2 mL to each plate of cells.
7. After ~4h refeed with normal media (can now contain antibiotics) and incubate overnight.

Purification of GST-S6K1

1. Treat cells for 30min with 100 nM Wortmannin to suppress endogenous S6K phosphorylation
2. Wash cells with D-PBS -/- twice (10 mL per plate)
3. Add 0.5 mL HNTG Buffer per plate and scrape
4. Collect scraped cells and incubate end over end in eppendorf tube for 30 min
5. Centrifuge in eppendorf centrifuge for 10 min at 14 000 RPM
6. Collect supernatant (save a lysate sample) into a 15 mL Falcon tube
7. Add 0.5 mL Glutatione-sepharose beads and incubate end over end for 1-2h.