Editing Pfu purification

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

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

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

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

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

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

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

Collect and pool Pfu fractions.

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

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

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

Combine Pfu fractions

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