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

ClpX Lysis Buffer (1L)

50mM TrisCl pH 8.0

100mM KCl

1mM MgCl2

10% Glycerol

1mM DTT

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