cells by centrifugation at 900 x g for 4 min.
- Resuspend in adequate volume of PBS for 5.5 x 106 to 6.0 x 106 cells/ml. Note: For example, one T-75 flask will yield approximately 7.5 x 106 cells 29.
- Keep cell suspension on ice until electroporation.
3. Preparation for Electroporation
- Pre-chill electroporation cuvettes (0.4 cm gap) on ice.
- Turn on electroporation apparatus and set the voltage to 0.3 kV.
NOTE: Capacitance and resistance were not adjustable settings on our electroporator (set at 10 μF and 600 Ω by the manufacturer).
- Fill recovery plates with NGM and equilibrate in humidified 95% air/5% CO2 atmosphere at 37° C.
- Place 400 µl of cell suspension in pre-chilled cuvette and add 20 µg of selected protein to cuvette (50 µg/ml).
- Flick 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.
- Dry outside of cuvette with paper towel or other wiper to avoid electrical arcing in the electroporator.
- Electroporate sample at 0.3 kV for 1.5-1.7 msec. Note: This was typical for this study.
- Immediately after electroporation flick cuvette gently ~10 times to mix thoroughly.
5. Plating of Cells
- Store cuvette with electroporated cells on ice until ready to place in recovery plates.
- For most downstream analyses, wash cells 1x with pre-warmed NGM to remove extraneous effector protein.
- Remove cells for analysis and suspend in 3-5 ml of NGM.
- Pellet cells by centrifugation at 900 x g for 4 min.
- Resuspend in adequate volume of NGM for desired plate size (e.g. 2 ml for 35 mm dish).
- Remove appropriate amount of cells for downstream analysis.
- Example 1: plate into glass bottom dishes for microscopy analysis.
- Example 2: plate into cell culture plastic for protein analysis such as affinity purifications.
- Allow cells to recover in equilibrated plates in humidified 95% air/5% CO2 atmosphere at 37° C for at least 4 hr.
6. Microscopy Analysis
6.1) Fixation/Immunofluorescence Staining
- Wash cells 1x with sterile PBS after 4 hr recovery period.
- 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).
- Wash 3x with sterile PBS.
- Permeabilize cells with 0.4% Triton X-100 in PBS for 15 min. For example, use 1 ml for a 35 mm diameter plate.
- Adjust length of permeabilization and strength of Triton X-100 according to epitope and location of 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.
- 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.
- Wash 3x with PBS.
- Incubate 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.
- Follow 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.
- Wash 4x with PBS.
- Incubate with appropriate fluorescently conjugated secondary antibody in antibody binding solution for 1 hr at room temperature, protected from light.
- For example, use Alexa 488 or Alexa 647 for 1 hr at room temperature.
- Follow manufacturer’s recommendations for antibody dilution. (e.g. about 1:1,000 for this study).
- Add other stains as needed, for example, 5 μM Wheat germ agglutinnin (WGA) conjugated to Alexa 647 or DAPI according to manufacturer’s recommendation, for 1 hr at room temperature.
- For example, use Alexa 488 or Alexa 647 for 1 hr at room temperature.
- Wash 5x with PBS and store at 4 °C, protected from light until ready to image.
6.2) Confocal Microscopy and Image Analysis
- Image samples on an inverted confocal microscope using a 63x oil immersion objective.
- Image green channel using the 488nm line of an argon laser, with band