Difference between revisions of "Purification of GST-HA-S6K"

Revision as of 17:13, 4 November 2009

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 cells into 10 new dishes in DMEM/FBS with no antibiotics.
2. Transefect with 90-95% confluent
3. Combine 240 ug DNA with 1.5 mL OptiMEM, and 600 uL Lipofectamine 2000 with 1.5 mL OptiMEM
4. After 5 minutes, combine the DNA solution with the Lipofectamine solution.
5. Wait 20 min, then add 3 mL to each plate of cells
6. After ~4h refeed with normal media (containing antibiotics)

Purification of GST-S6K1

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