Difference between revisions of "Purification of GST-HA-S6K"
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#Collect supernatant (save a lysate sample) into a 15 mL Falcon tube | #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. | #Add 0.5 mL Glutatione-sepharose beads and incubate end over end for 1-2h. | ||
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| + | [[Category: Protein Purification]] | ||
| + | [[Category: mTOR]] | ||
| + | [[Category: Kinase Assay]] | ||
Revision as of 15:32, 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
- Split cells into 10 new dishes in DMEM/FBS with no antibiotics.
- Transefect with 90-95% confluent
- Combine 240 ug DNA with 1.5 mL OptiMEM, and 600 uL Lipofectamine 2000 with 1.5 mL OptiMEM
- After 5 minutes, combine the DNA solution with the Lipofectamine solution.
- Wait 20 min, then add 3 mL to each plate of cells
- After ~4h refeed with normal media (containing antibiotics)
Purification of GST-S6K1
- 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.
