Editing Protein Production using Baculovirus (section)

== Samll Scale Expression<br>  ==

<br>Use the following procedure for harvesting recombinant baculovirus infected insect cells to analyze expression of your recombinant protein of interest in a 24-well format 

1. Seed 6 X 10<sup>5</sup> Sf9 or Sf21 cells per well in a 24-well plate. Let cells attach for at least 30 minutes.<br>2. Remove the media and rinse the cells once with fresh growth media. Replace with 300 μl of fresh media.<br>3. Add baculoviral stock to each well at the desired MOI. Include the appropriate controls (e.g., mock-infected (uninfected) cells, pFastBac<br> positive control baculovirus, previously characterized recombinant baculoviruses).<br>4. Incubate cells in a 27 °C, humidified incubator.<br>5. Harvest cells (or media, if the recombinant protein is secreted) at the appropriate time (i.e., 24, 48, 72, 96 hours post-infection). If harvesting cells, remove the media and rinse the cells once with serum-free medium.<br>6. Lyse the cells with 400 μl of 1X SDS-PAGE Buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS).<br>7. Freeze samples at –20 °C or boil samples for at least 3 minutes.<br>8. Run SDS-PAGE and detect recombinant protein by western blot