escence 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 bandwidth emission between 492-542 nm.
- Image red channel using a 633 nm diode laser, with bandwidth emission between 640-718 nm.
- Image blue channel using a 405 nm diode laser, with bandwidth emission between 407-453 nm.
- Ensure the multiple channel z-stacks cover the entirety of the cellular volume.
- Process images with appropriate image processing software.
7. Affinity Purification
- Wash electroporated cells twice with 4 °C PBS after 4 hr recovery period.
- Lyse with ~1.0 ml of lysis buffer (1% Triton X-100 with protease inhibitor cocktail and phosphatase inhibitor in PBS) on ice. Use inhibitors according to manufacturer instructions. Note: For example, both the phosphatase and protease inhibitors used in this study were supplied at 100x, but other formulations should work equally well.
- Promptly scrape cells with rubber policeman and collect into conical tubes.
- Lyse by vigorous vortexing and sonication. Note: Other lysis methods may work equally as well, but efficiency will need to be empirically determined as to the suitability of assay requirements.
- Sonicate 3 x 30 sec with intermittent vortexing.
- Centrifuge at 10,000 x g for 10 min at 4 °C to collect cellular debris and insoluble aggregates; save the supernatant.
- Combine equal volumes (~1.0 ml) of electroporated cell lysate with 50 µl of Streptavidin agarose resin suspension at 4 °C overnight with end-over-end rotation. Note: This resin captures the electroporated protein (and associated complexes) via its streptavidin-binding peptide affinity tag.
- Centrifuge at 2,500 x g for 2 min and discard supernatant.
- Wash twice with 40 bed volumes (~1 ml) PBS.
- Add 30 µl 4x LDS loading buffer (141 mM Tris base, 2% LDS, 10% glycerol, 0.51 mM EDTA, 0.22 mM SERVA Blue G, 0.175 mM phenol red, pH 8.5), 20 µl diH2O, and 1 µl of 0.5 M tris(2-carboxyethyl)phosphine (TCEP), allowing for some volume to remain in beads.
- Heat at 95 °C for 10 min and cool on ice.
- Spin >10,000 x g at 4 °C for 5 min.
- Collect supernatant.
- Perform a western blot using appropria