Purification of GST-HA-S6K

Revision as of 15:32, 4 November 2009 by Davebridges (Talk | contribs) (initial page)

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Revision as of 15:32, 4 November 2009 by Davebridges (Talk | contribs) (initial page)

(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

Materials

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.