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, DAPI) in conjunction with anti-fade reagent||Any||N/A|
|RAW 264.7 Cell line||ATCC||TIB-71|
|Primary Antibody Against Target of Interest||Any||N/A|
|Secondary Antibody Conjugated to Fluorophore||Any||N/A|
|Phosphate Buffered Saline||Any||N/A||Chill to 4 °C|
|Sterile Phosphate Buffered Saline||Any||N/A||Warm to 37 °C|
|Streptavidin Agarose Resin Suspension||Pierce||20353|
|Table Top Centrifuge Capable of Accepting Conical Tubes (swinging bucket preferred)||Any||N/A|
|Triton X-100||Sigma-Aldrich||T8585||Irritant, Toxic|
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