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-HCl at pH 7.5

200mM KCl

25mM MgCl2

0.1mM EDTA

1mM DTT

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
• 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.
• 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