Sauer:His6 ClpX purification

Materials Needed

 * 1000x AMP: 100mg/mL in 70% Ethanol
 * 2 x 1L TB
 * 1M ZnSO4
 * 1M IPTG
 * His-tagged ClpX strain: SJ01
 * LB

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

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