Editing Baculovirus Expression (section)

=== <br>C) Metal affinity-purify the protein of interest (intracellular proteins). ===

<br>1) Wash the infected cells once or twice with ice cold TBS (50 mM Tris, 100 mM NaCl; pH 8.0).

<br>2) Lyse the cells in one of two ways:<br>a) Resuspend pellet in a minimal volume of ice-cold dialysis buffer; freeze-thaw twice.<br>b) Resuspend pellet in a minimal volume of TBS + 1% NP40); incubate for 10 min on ice.

<br>3) Shear DNA by tituration with an 18-gauge needle until the solution is no longer viscous.

<br>4) Centrifuge for 15 min at ~10,000 xg at 4oC.

<br>5) Harvest the supernatant and use for metal affinity chromatography as described above (D2).