A quick way to screen bacterial colonies for those expressing a GST-fusion protein from a pGEX vector is to monitor whole cell lysates on SDS-PAGE.
  1. Restreak individual colonies on sectors of LB/amp plates.  Be sure to include a sector of cells containing the original pGEX plasmid as this will serve as a positive control, which expresses GST.
  2. Incubate plates at 37 oC for overnight or until growth is visible.
  3. Inoculate sterile test tubes containing 2 mls of LB/amp with small portions of cells from the overnight plate.
  4. Grow the cells at 37oC for 3 hours or until the clutures are cloudy.
  5. Transfer 500 ul of cells from each tube to a 1.5 ml microfuge tube and place on ice.  Lysates from these cells will serve as uninduced controls.
  6. Add 2 ul of 100 mM IPTG to the remaining 1.5 ml of cells and allow the cells to grow for 2 more hours.
  7. Transfer 500 ul of IPTG-induced cells to a 1.5 ml microfuge and place on ice.
  8. Collect the induced and uninduced cells by centrifuging the cells for 20 seconds at top speed in a microfuge.
  9. Remove the supernatant and resuspend the cells in 50 ul of phosphate buffered saline (PBS).
  10. Add 50 ul of 2X SDS-PAGE sample buffer.  Store whole cell lysates at -70 oC until you are ready to analyze 10 ul of each lysate on a 12% SDS-polyacrylamide gel.