Sauer:His6 ClpX purification

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SAUER LAB

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Contents

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
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