uired 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, multiple proteins may be introduced simultaneously, or drugs and other small molecules can be delivered alongside proteins. Most important for obtaining a functional understanding of the effect of the introduced proteins, protein electroporation could be coupled with assays for protein function and interactions, such as affinity purification, western blots against known targets, whole-cell expression analysis, and microscopy to determine subcellular localization of the introduced protein. Hence, this method has great potential as a tool for elucidating bacterial pathogen biology alongside other cellular and molecular biology techniques.
The authors have nothing to disclose.
This work was supported by NIGMS, National Institutes of Health (GM094623). Significant portions of this work were performed in the Environmental Molecular Sciences Laboratory, a DOE/BER national scientific user facility located at the Pacific Northwest National Laboratory (PNNL). PNNL is operated for the DOE by Battelle under Contract DE-AC05-76RLO1830.
|0.25% Trypsin-EDTA Solution||Cellgro||25-050-Cl|
|0.4 cm Gap-disposable electroporation cuvettes||Bio-Rad||165-2088|
|100% Methanol||Any||N/A||Flammable, Toxic|
|Bovine Serum Albumin (BSA)||Sigma-Aldrich||A4919|
|Cell Counting Apparatus - Hemocytometer or Coulter Counter||Beckman Coulter||Model Z1|
|Cell Culture Incubator||Any||N/A||Humidified 95% air/5% CO2 atmosphere at 37 °C|
|Cell Culture Plastic||Any||N/A||Cell culture flasks/plates, pipets, tubes, rubber policeman|
|Dulbecco's Modification of Eagle’s Medium (DMEM)||Cellgro||10-013||Warm to 37 °C|
|Electroporator||Bio-Rad||E. coli Pulser|
|Fetal Bovine Serum (FBS)||Cellgro||35-016-CV|
|Fluorescent confocal microscope||Ziess||Model LSM 710|
|Glass Bottom Dishes for Microscopy||Wilco Wells||HBSt-3522|
|HALT Protease Inhibitor Cocktail||Pierce||78430||Corrosive, Toxic|
|HeLa Cell Line||ATCC||ATCC CCL-2|
|LDS 4X Loading Buffer||Invitrogen||NP0007|
|Minimal Essential Medium (MEM)||Cellgro||10-010||Warm to 37 °C|
|Other fluorescent stains (WGA