. 1 x 106 cells), though the best cell number will vary with cell size and type. Another critical step in the experiment was to allow cells time to recover and reattach to dishes; this was necessary because adherent cells were used in this study. A recovery period should be less important for suspension cells, although it may be necessary to give proteins time for proper intracellular trafficking, protein-protein interactions, or enzymatic activity, depending on the nature of the intended study. Since delivered proteins were observed inside cells for up to 96 hr, there is much room for adjustment to the incubation period prior to microscopy visualization or cellular assay.
Several variables in this protocol may be optimized for different cell types, protein targets, or downstream analysis schemes. The amount of protein to be electroporated can be fine-tuned for the desired intracellular concentration. For visualization of protein localization, as little as 1 µg can be used. For functional studies, higher starting amounts of proteins may be needed. In addition, the amount of time for post-electroporation cell recovery can be shorted or extended. Labile protein targets or fast downstream processes would require a shorter incubation time. Also, the amount of cells plated can be adjusted beyond the suggestions here. Cells may be plated more sparsely if the introduced protein is to be monitored by microscopy, or plated more densely if the electroporated protein is to be recovered for applications such as western blots.
While electroporation is a simple and straightforward method for introducing proteins into living cells, there are some limitations to the technique. The method requires highly pure proteins in microgram amounts. As little as 1 µg was electroporated and visualized with confocal microscopy, but higher starting amounts of protein gave better visual results. While protein function was not assessed at all concentrations after electroporation, protein concentrations equal to or greater than 50 μg/ml were required for adequate signal for western blot. Also, due to the size constraints of the electroporation cuvettes, scaling up may require processing of multiple cuvettes of cells. However, considering that the electroporation process takes mere seconds once cells are prepared, parallel processing requires little extra time and effort.
If the introduced protein is to be visualized by western blot, microscopy, or used for affinity purification, the protein should have a tag 35 for affinity purification or an available antibody for detection. However, for unknown reasons, some interactions known from the literature or other biochemical methods (data not shown) were not able to be recapitulated after electroporation. It may be that the host recognizes some electroporated proteins as foreign and quickly marks them for proteasome degradation.
Electroporation holds several advantages over other existing methods for protein delivery. Compared to microinjection, electroporation is much faster and simpler, and a large number of cells can be treated at once with almost 100 percent viability for the tested conditions. Electroporation is also cheaper than protein transfection with commercially available reagents. DNA transfections are often used to study bacterial effector proteins in host cells; however, this causes the bacterial proteins to be synthesized non-ideally by the host. On the other hand, electroporation of bacterial proteins removes the reliance on mammalian protein expression machinery, allowing for a better representation of the infectious process where effector proteins are expressed by the bacteria.
Several applications could use protein electroporation as a method in the study of bacterial-host interactions. First, primary cells that are often difficult to transfect may be more amenable to electroporation for protein delivery. This will allow for the study of effector function in more biologically relevant cell types. Electroporation also gives the option of multiplexing proteins and/or treatments. For example, 