Martin Humphries
LAB

Protocols - GST-PAK and GST-GGA3 beads production


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Auto-induction media


  1. 10% (w/v) tryptone
  2. 5% (w/v) yeast extract
  3. 25mM Na2HPO4
  4. 25mM KH2PO4
  5. 50mM NH4Cl
  6. 5mM Na2SO4
  7. 2mM MgSO4
  8. 2mM CaCl2
  9. 0.5% (v/v) glycerol
  10. 0.05% (w/v) glucose
  11. 0.2% (w/v) lactose
  12. 25mg.ml-1 ampicillin


  13. Protein production


    1. Growth JM109 E.coli cells expressing the ampicillin resistant DNA of interest for 8 hours at 37°C, 220rpm.
    2. Resuspend JM109 culture in autoinduction media and incubat overnight at 37°C, 220rpm
    3. Harvest cells by centrifugation (5,000rpm JlA10.5 rotor, Beckman, 20min)
    4. resuspend cells in TBS containing complete mini protease inhibitor cocktail tablet (Roche)
    5. lyse cells for 30min at RT using bug buster (Novagen, Nottingham, UK) supplemented with 0.05mg.ml-1 RNaseA and 0.05 mg.ml-1 Dnase1
    6. Clarify cell lysates by centrifugation at 18,000rpm (JA25.5 rotor, Beckman) for 20min
    7. Collect the soluble cell lysate and incubate it with pre-washed Glutatione Sepharose beads (GE Healthcare, Slough UK) for 1h at RT, to allow protein binding
    8. Wash 3 times the beads with PBS
    9. Resuspend the beads in PBS in a 1:1 ratio, ready for use
    10. Resuspend 10uL of beads in sample buffer, and check by SDS-Page followed by comassie staining, the presence of the GST protein


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