Introduction of Bacterial Effector Proteins into Mammalian Cells by Electroporation

1. Electroporation

1. Place 400 µl of cell suspension in a pre-chilled cuvette and add 20 µg of selected protein to the cuvette (50 µg/ml).
2. Flick the cuvette gently ~10 times to mix without damaging cells. Note: The cuvette may also be inverted several times to mix thoroughly, but do not pipet up and down or vortex to avoid damaging cells.
3. Dry the outside of the cuvette with a paper towel or other wiper to avoid electrical arcing in the electroporator.
4. Electroporate sample at 0.3 kV for 1.5-1.7 msec. Note: This was typical for this study.
5. Immediately after electroporation, flick the cuvette gently ~10 times to mix thoroughly.

2. Plating of Cells

1. Store the cuvette with electroporated cells on ice until ready to place in recovery plates.
2. For most downstream analyses, wash cells 1x with pre-warmed NGM to remove extraneous effector protein.
   1. Remove cells for analysis and suspend in 3-5 ml of NGM.
   2. Pellet cells by centrifugation at 900 x g for 4 min.
   3. Resuspend in an adequate volume of NGM for the desired plate size (e.g., 2 ml for a 35 mm dish).
3. Remove the appropriate amount of cells for downstream analysis.
   1. Example 1: Plate into glass bottom dishes for microscopy analysis.
   2. Example 2: Plate into cell culture plastic for protein analysis such as affinity purifications.
4. Allow cells to recover in equilibrated plates in a humidified 95% air/5% CO₂ atmosphere at 37° C for at least 4 hr.

3. Microscopy Analysis

1. Fixation/Immunofluorescence Staining
   1. Wash cells 1x with sterile PBS after a 4-hour recovery period.
   2. Fix cells in 100% methanol for 2 min at room temperature. Use enough methanol to completely cover cells (e.g. 2 ml for 35 mm dish).
   3. Wash 3x with sterile PBS.
   4. Permeabilize cells with 0.4% Triton X-100 in PBS for 15 min. For example, use 1 ml for a 35 mm diameter plate.
      1. Adjust the length of permeabilization and strength of Triton X-100 according to the epitope and location of the target protein. Note: Best results will need to be empirically determined for each target, however, the above conditions should be adequate for most cytosolic targets.
   5. Block with 5% Bovine Serum Albumin (BSA) in PBS for 1 hr at RT. For example, use 1 ml for a 35 mm diameter plate.
   6. Wash 3x with PBS.
   7. Incubate the primary antibody in antibody binding solution (0.1% Triton X-100 and 1% BSA in PBS) overnight at 4 °C with gentle rocking. For example, use 0.5 ml for a 35 mm diameter plate.
      1. Follow the manufacturer's recommendations for antibody dilution. Note: For example, the streptavidin-binding peptide tag (SBP-tag) antibody was used at 1:1,000 dilution, while the PKN1 antibody was used at 1:200 dilution.
   8. Wash 4x with PBS.
   9. Incubate with appropriate fluorescently conjugated secondary antibody in antibody binding solution for 1 hr at room temperature, protected from light.
      1. For example, use Alexa 488 or Alexa 647 for 1 hour at room temperature.
         1. Follow the manufacturer's recommendations for antibody dilution. (e.g., about 1:1,000 for this study).
      2. Add other stains as needed, for example, 5 μM Wheat germ agglutinin (WGA) conjugated to Alexa 647 or DAPI according to manufacturer's recommendation, for 1 hr at room temperature.
   10. Wash 5x with PBS and store at 4 °C, protected from light until ready to image.