Jhdavis::ClpPHisPrep

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Current revision (16:08, 26 January 2009) (view source)
 
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'''Protocol'''
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E. coli ClpP protein with a C-terminal His6 tag was expressed in E. coli strain DH5α/pYK133/pRep4.  
E. coli ClpP protein with a C-terminal His6 tag was expressed in E. coli strain DH5α/pYK133/pRep4.  
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Fractions containing ClpP were pooled, dialyzed against Clp buffer at 4°C, and stored at −80°C. Concentrations of ClpP14  and ClpX6  were determined by UV absorbance (e = 125160 M-1 cm-1)
Fractions containing ClpP were pooled, dialyzed against Clp buffer at 4°C, and stored at −80°C. Concentrations of ClpP14  and ClpX6  were determined by UV absorbance (e = 125160 M-1 cm-1)
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!!Buffers:!!
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'''Buffers:'''
#PD buffer: contains 25 mM HEPES-KOH (pH 7.6), 5 mM KCl, 5 mM MgCl2, 0.032% NP-40, 10% glycerol, 5 mM ATP, and an ATP-regenerating system consisting of 16 mM creatine phosphate and 0.32 mg/ml creatine kinase.  
#PD buffer: contains 25 mM HEPES-KOH (pH 7.6), 5 mM KCl, 5 mM MgCl2, 0.032% NP-40, 10% glycerol, 5 mM ATP, and an ATP-regenerating system consisting of 16 mM creatine phosphate and 0.32 mg/ml creatine kinase.  
#Clp buffer:  50 mM Tris-HCl (pH 7.5), 200 mM KCl, 25 mM MgCl2, 1 mM DTT, 0.1 mM EDTA, and 10% glycerol.  
#Clp buffer:  50 mM Tris-HCl (pH 7.5), 200 mM KCl, 25 mM MgCl2, 1 mM DTT, 0.1 mM EDTA, and 10% glycerol.  

Current revision

Protocol

E. coli ClpP protein with a C-terminal His6 tag was expressed in E. coli strain DH5α/pYK133/pRep4.

Cultures were grown at 30°C to an OD600 of 0.5 in LB with antibiotics, and IPTG was added to 0.5 mM.

Cells were harvested by centrifugation 2–3 hr later and resuspended in 3 ml of S buffer per gram of cells.

Following sonication, the lysate was centrifuged for 20 min at 17,000 × g, and the supernatant (about 30 ml) was added to 1.5 ml nickel-NTA resin (Qiagen), preequilibrated with S buffer.

After mixing for 1 hr at 4°C, the resin was packed into a column and washed with 200 ml S buffer and 100 ml W20 buffer.

Protein was eluted with W500 buffer, and fractions containing ClpP were pooled and

desalted into Q50 buffer.

This material was loaded onto a MonoQ HR5/5 FPLC column (Pharmacia) and eluted with a 20 ml gradient from Q50 to Q1000 buffer.

Fractions containing ClpP were pooled, dialyzed against Clp buffer at 4°C, and stored at −80°C. Concentrations of ClpP14 and ClpX6 were determined by UV absorbance (e = 125160 M-1 cm-1)

Buffers:

  1. PD buffer: contains 25 mM HEPES-KOH (pH 7.6), 5 mM KCl, 5 mM MgCl2, 0.032% NP-40, 10% glycerol, 5 mM ATP, and an ATP-regenerating system consisting of 16 mM creatine phosphate and 0.32 mg/ml creatine kinase.
  2. Clp buffer: 50 mM Tris-HCl (pH 7.5), 200 mM KCl, 25 mM MgCl2, 1 mM DTT, 0.1 mM EDTA, and 10% glycerol.
  3. S buffer: 50 mM Na-phosphate (pH 8.0), 1 M NaCl, 5 mM imidazole, and 10% glycerol.
  4. W20 buffer: 50 mM Na-phosphate (pH 8.0), 1 M NaCl, 10% glycerol, and 20 mM imidazole;
  5. W500 buffer is the same but contains 500 mM imidazole.
  6. Q50 buffer: 50 mM Tris (pH 8.0), 5 mM DTT, 10 mM MgCl2, 10% glycerol, and 50 mM KCl;
  7. Q1000 buffer is the same but contains 1 M KCl.
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