width 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 appropriate antibodies Note: Here, anti-PKN1 primary antibody is used.
As an initial proof of concept, purified green fluorescent protein was successfully introduced into mammalian cells using electroporation. GFP, an approximate 27 kD a molecular weight protein is commonly introduced into mammalian cells (normally expressed from plasmid DNA) as a molecular biology tool without significant cellular toxicity. HeLa cells were incubated (Figure 1A) or electroporated (Figure 1B) with 25 µg/ml GFP, followed by immunofluorescence confocal microscopy to check for fluorescent GFP signal. To demonstrate that the GFP was inside the cellular cytosol, the cells were also stained with wheat germ agglutinin (WGA) to delineate the cytosolic boundary (red) as well as a nucleic acid stain, DAPI (blue) to indicate the nucleus. Intracellular GFP signal was observed only upon electroporation, while no intracellular protein was observed in cells incubated with GFP. Similar results were observed with RAW264.7 mouse macrophage-like cells (Figure 1C and 1D). Note that WGA is a lectin that preferentially binds to residues in the plasma membrane and thus can exhibit punctate staining based on the length of incubation. Compare Figure 1A and Figure 2A, where Figure 2A was incubated for a longer duration than Figure 1A.
Electroporation was then extended to a tagged, purified Salmonella effector, GtgE. GtgE, a known virulence factor 30, was discovered by our group to be secreted into host cells 31, and was recently shown to be a cysteine protease 32. HeLa cells were incubated or electroporated with 50 µg/ml GtgE. For immunofluorescence analysis, the cells were stained with an antibody against the streptavidin-binding peptide affinity tag on GtgE. The cells were also stained with WGA and DAPI. In incubated HeLa cells (Figure 2A), there is no intracellular GtgE as visualized by a lack of green fluorescent foci. In contrast, electroporated HeLa cells (Figure 2B) show significant fluorescent intracellular signal indicating effector protein had entered the