Sauer:GST-sFtsH purification: Difference between revisions
(New page: {{Sauer lab sidebar}} ===GST-sFtsH purification=== 1) Inoculate overnight from glycerol stock. 2) Wash overnight growth (spin down, resuspend, repeat) before inoculating 2x1L TB/Amp 3) ...) |
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===GST-sFtsH purification=== | ===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: | Lysis Buffer: | ||
50mM TrisHCl pH 8 | 50mM TrisHCl pH 8 | ||
Line 26: | Line 28: | ||
1Mm 2mM DTT | 1Mm 2mM DTT | ||
==Purification== | |||
# Lyse cells by chemical lysis in the presence of protease inhibitor cocktail. | # Lyse cells by chemical lysis in the presence of protease inhibitor cocktail. | ||
# Centrifuge. | # Centrifuge. |
Revision as of 14:31, 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
4) Ammonium Sulfate cut- add sat AmSO4 to 60%. (make sure to add slowly, with stirring, in the cold room) Let incubate 1h + 5) Centrifuge, 20m by 12krpm 6) Resuspend in lysis buffer 7) PD10 to remove excess salt 8) Add Glutathione Agarose resin. Let incubate 30m. 9) Wash column with 10 volumes lysis buffer 10) Elute 2x with Glutathion Ag Elution Buffer 11) Buffer Exchange eluted protein to remove excess glutathione 12) Precision Protease (40uL per L starting culture) 40h 13) Incubate with Glutathione Agarose. Collect flow through + 1-2 CV of wash. 14) Concentrate 15) 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)