GST-PAK and GST-GGA3 beads production
GST-PAK and GST-GGA3 beads production
Auto-induction media
- 10% (w/v) tryptone
- 5% (w/v) yeast extract
- 25mM Na2HPO4
- 25mM KH2PO4
- 50mM NH4Cl
- 5mM Na2SO4
- 2mM MgSO4
- 2mM CaCl2
- 0.5% (v/v) glycerol
- 0.05% (w/v) glucose
- 0.2% (w/v) lactose
- 25mg.ml-1 ampicillin
Protein production
- Growth JM109 E.coli cells expressing the ampicillin resistant DNA of interest for 8 hours at 37°C, 220rpm.
- Resuspend JM109 culture in autoinduction media and incubat overnight at 37°C, 220rpm
- Harvest cells by centrifugation (5,000rpm JlA10.5 rotor, Beckman, 20min)
- resuspend cells in TBS containing complete mini protease inhibitor cocktail tablet (Roche)
- 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
- Clarify cell lysates by centrifugation at 18,000rpm (JA25.5 rotor, Beckman) for 20min
- 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
- Wash 3 times the beads with PBS
- Resuspend the beads in PBS in a 1:1 ratio, ready for use
- Resuspend 10uL of beads in sample buffer, and check by SDS-Page followed by comassie staining, the presence of the GST protein
GST-PAK and GST-GGA3 beads production – right hand column
Other protocols
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