Sauer:His6 ClpX purification: Difference between revisions
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===Materials Needed=== | ===Materials Needed=== | ||
1000x AMP: 100mg/mL in 70% Ethanol | *1000x AMP: 100mg/mL in 70% Ethanol | ||
*2 x 1L TB | |||
2 x 1L TB | *1M ZnSO4 | ||
*1M IPTG | |||
1M ZnSO4 | *His-tagged ClpX strain: SJ01 | ||
*LB | |||
1M IPTG | |||
His-tagged ClpX strain: SJ01 | |||
LB | |||
===Protocol=== | ===Protocol=== | ||
| Line 44: | Line 39: | ||
===Materials Needed=== | ===Materials Needed=== | ||
Clp Buffer: | ====Clp Buffer:==== | ||
*50mM HEPES-KOH at pH 7.5 | |||
*200mM KCl | |||
*25mM MgCl2 | |||
*0.1mM EDTA | |||
*10% glycerol | |||
====Lysis Buffer (1L)==== | |||
*50 mM NaH<sub>2</sub>PO<sub>4</sub> pH 8.0 | |||
*300mM NaCl | |||
*100mM KCl | |||
*10mM imidazole | |||
*10% Glycerol | |||
*1mM DTT | |||
====Wash buffer ==== | |||
*50 mM NaH<sub>2</sub>PO<sub>4</sub> | |||
*500 mM NaCl | |||
*20 mM imidazole | |||
Adjust to pH 8 | |||
====Elution buffer==== | |||
*50 mM NaH<sub>2</sub>PO<sub>4</sub> | |||
*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=== | |||
10 | *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 | |||
Latest revision as of 08:46, 7 November 2007
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
