Paul.xu at 22:12, 21 December 2017

2017-12-21T22:12:42Z

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Grow cells, induce with IPTG, spin down cells.

To 50 g cell paste add 200 ml of '''Buffer A''':

50 mM Tris-HCl, 500 mM NaCl, pH 8.0, 1 mM beta mercaptoethanol

Lyse by freeze and thaw

Incubate at 70°C for 1 hour

Cool on ice for 15 min

Homogenize

Spin at 25,000 rpm for 30 min.

Load supernatant to a Ni column, pre-equilibrated with '''Buffer A''' , 500 mM NaCl; 50 mM Tris-HCl (pH 7,5), 20 mM Imidazole

Wash column with  '''Buffer A''' , 500 mM NaCl; 50 mM Tris-HCl (pH 7,5), 20 mM Imidazole

Wash column once 1 CV with '''Buffer C''': 50 mM Tris-HCl (pH 7,5)

Elute protein with '''Buffer B''': 50 mM NaCl; 50 mM Tris-HCl (pH 7,5), 150 mM Imidazole

Dilute eluted enzyme with  '''Buffer D''': 50 mM Tris-HCl, pH 8.0 

Load onto Q-sepharose  HP column. Wash with '''Buffer D''': 50 mM Tris-HCl, pH 8.0.

Linear gradient from  '''Buffer D to Buffer E''': 50 mM Tris-HCl, pH 8.0, 600 mM NaCl to elute Pfu.

Collect and pool Pfu fractions.

Dilute combined enzyme with '''Buffer D''': 50 mM Tris-HCl, pH 8.0.

Load diluted enzyme to a phosphocellulose or Heparin column (2.5×40 cm)  or at room temperature. Wash with '''Buffer D '''until the optical density measured at an absorbance of 280 nm drop  to baseline.

Elute Pfu with linear gradient '''Buffer D '''to  '''Buffer F''': 1M  KCl; 50 mM Tris-HCl, pH 8,0.

Combine Pfu fractions

Run SDS-PAGE to check purity.
[[Category:Protocols]]

Pfu purification - Revision history

Paul.xu at 22:12, 21 December 2017