Sauer:His6 ClpX purification
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Expression
Materials Needed
- 1000x AMP: 100mg/mL in 70% Ethanol
- 2 x 1L TB
- 1M ZnSO4
- 1M IPTG
- His-tagged ClpX strain: SJ01
- LB
Protocol
Day One
- 1. Innoculate 25mL of LB-AMP with His-tagged ClpX strain.
- 2. Warn 2x 1L of TB in incubator or warm room at 37 overnight
Day Two
- 1. Add 1mL of 1000x AMP to each of the TB Flasks.
- 2. Innoculate each flask with 10mL of overnight culture
- 3. Grow at 37 C until OD is ~1-1.2 (should take ~>3hrs.)
- 4. Drop temperature of incubator to <RT
- 5. Add 15uL of ZnSO4
- 6. At OD of 1.7-2.0, induce with 0.25mM IPTG (250uL). Make sure flask is cooled to RT)
- 7. After 2-3 hrs. move to bottles and spin down in J6 at 4K/
- 8. Decant Supernatant. Immediately Freeze at -80
Purification
Materials Needed
Clp Buffer:
- 50mM HEPES-KOH at pH 7.5
- 200mM KCl
- 25mM MgCl2
- 0.1mM EDTA
- 10% glycerol
Lysis Buffer (1L)
- 50 mM NaH2PO4 pH 8.0
- 300mM NaCl
- 100mM KCl
- 10mM imidazole
- 10% Glycerol
- 1mM DTT
Wash buffer
- 50 mM NaH2PO4
- 500 mM NaCl
- 20 mM imidazole
Adjust to pH 8
Elution buffer
- 50 mM NaH2PO4
- 500 mM NaCl
- 250 mM imidazole
Adjust to pH 8
Q Loading Buffer 1L
- 50mM HEPES-KOH pH 7.6
- 100mM KCl
- 1mM MgCl2
- 10% Glycerol
- 1mM DTT
Q Elution Buffer 1L
- 50mM HEPES-KOH pH 7.6
- 1M KCl
- 1mM MgCl2
- 10% glyercol
- 1mM DTT
Purification
- 1. Thaw with lysis buffer. Remember to add protease inhibitor.
- 2. Lyse via method of your choice
- 3. Spin down 50m. at 15k rpm
Ni-NTA
- 4. Incubate supernatant with prewashed Ni-NTA slurry for ~30m to 1hr.
- 5. Pour into column. Wash with ~20mL NiNTA wash buffer.
- 6. Elute with ~15mL N.E. buffer, collecting 1mL fractions
- 7. Identify best fractions by Bradford Assay
- 8. Pool best fractions, buffer exchange (PD10) into Q loading buffer.
MonoQ
- 9. MonoQ Column, 20 min. (or longer) gradient to 100% Q Elution.
- 10. Concentrate as needed. Buffer exchange into Clp buffer
- 11. Store at -80
