Sauer:GST-sFtsH purification: Difference between revisions
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==Buffers== | ==Buffers== | ||
Lysis Buffer: | |||
*50mM TrisHCl pH 8 | *50mM TrisHCl pH 8 | ||
*1mM EDTA | *1mM EDTA | ||
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# Centrifuge. | # Centrifuge. | ||
# Resuspend in lysis buffer | # Resuspend in lysis buffer | ||
# Ammonium Sulfate cut- add sat AmSO4 to 60%. (make sure to add slowly, with stirring, in the cold room) | # Ammonium Sulfate cut- add sat AmSO4 to 60%. (make sure to add slowly, with stirring, in the cold room) Let incubate 1h + | ||
# Centrifuge, 20m by 12krpm | # Centrifuge, 20m by 12krpm | ||
# Resuspend in lysis buffer | # Resuspend in lysis buffer | ||
Latest revision as of 14:33, 27 July 2009
GST-sFtsH purification
Expression
- Inoculate overnight from glycerol stock.
- Wash overnight growth (spin down, resuspend, repeat) before inoculating 2x1L TB/Amp
- Grow to OD 1, induce with 0.5mM IPTG.
- Let grow ~2h. Store at -80.
Buffers
Lysis Buffer:
- 50mM TrisHCl pH 8
- 1mM EDTA
- 1mM DTT
Glutathione Ag Elution Buffer:
- 50mM Tris HCl
- 10mM reduced glutathione
- 400mM KCl
Generic buffer (GB) for this purification
- 50mM Tris-HCl pH 8
- 150mM KCl
- 10mm MgCl2
- 10uM Zinc acetate
- 1Mm EDTA
- 1Mm 2mM DTT
Purification
- Lyse cells by chemical lysis in the presence of protease inhibitor cocktail.
- Centrifuge.
- Resuspend in lysis buffer
- Ammonium Sulfate cut- add sat AmSO4 to 60%. (make sure to add slowly, with stirring, in the cold room) Let incubate 1h +
- Centrifuge, 20m by 12krpm
- Resuspend in lysis buffer
- PD10 to remove excess salt
- Add Glutathione Agarose resin. Let incubate 30m.
- Wash column with 10 volumes lysis buffer
- Elute 2x with Glutathion Ag Elution Buffer
- Buffer Exchange eluted protein to remove excess glutathione
- Precision Protease (40uL per L starting culture) 40h
- Incubate with Glutathione Agarose.
- Collect flow through + 1-2 CV of wash.
- Concentrate
- Superdex 300 column
Concentrate/buffer exchange in GB
Elute Glutathione Agarose column with EB- keep to ensure success in cleavage
S300 size-exclusion column (S200 was used here in the published protocol, but the protein if hexameric should be in the exclusion volume with the S200)
