Electroporation of Functional Bacterial Effectors into Mammalian Cells

The overall goal of the following experiment is to introduce exogenous, potentially cytotoxic proteins such as bacterial effectors into the mammalian cells to study their effects and function. This is achieved by first growing mammalian cells via standard culture condition to obtain suitable host like cells in which to introduce the purified protein of interest. As a second step electroporation of the cultured host cells in the presence of a purified bacterial effector is conducted, which allows the protein to enter and interact with the host cells.

Next, visualization or some other functional assay is performed in order to assess the subcellular localization and the functionality of the effector protein. The results based on confocal microscopy, immunochemical methods, or other analysis techniques show the localization of the functional bacterial effectors to their expected subcellular locations and the binding to their known interaction partners. The main advantage of this technique over existing methods like transection or transduction, is that this protocol is a fast, simple, and inexpensive approach to overcome potential cytotoxicity.

This method can help answer key questions in the pathogen biology field, such as discovery or validation of the functions of bacterial effector proteins. Though this method was used to provide insight into bacterial effector proteins, it can also be applied to other proteins or small molecules. Visualizing result is critical.

Since colocalization with known cellular features, verify the expected subcellular association of the Electroporated protein To begin this procedure. Transfer 400 microliters of cell suspension to a pre chilled vete. Then add 20 micrograms of selected protein to reach a final concentration of 50 micrograms per milliliter.

Flick the vete gently about 10 times for mixing and to avoid damaging the cells. After that, dry the outside of the Q Vet with a paper towel to avoid electrical arcing in the electro. Place the sample in the electro and set it to 0.3 kilovolts for 1.5 to 1.7 milliseconds immediately after electroporation.

Flick the qve gently about 10 times to mix the sample thoroughly before proceeding to either fixation and immunofluorescent staining or affinity purification. After four hours of recovery from electroporation, wash the cells with sterile PBS. Then fix the cells in 100%methanol for two minutes at room temperature.

Subsequently, wash the cells with sterile PB S3 more times. Perme the cells with 0.4%tritton X 100 in PBS for 15 minutes. Adjust the length of permeable and the strength of Triton X 100 according to the epitope and location of the target protein.

Then block the cells with 5%BSA in PBS for one hour at room temperature.Afterward. Wash them three times with PBS. Next, incubate them with the primary antibody in the antibody binding solution overnight at four degrees Celsius with gentle rocking the next day.

Wash the cells four more times with PBS. Then incubate them with the appropriate fluorescently conjugated secondary antibody in the antibody binding solution. Add other stains as needed, such as five micromolar wheat germ agglutinin or WGA conjugated to Alexa 6 47 or DPI for one hour at room temperature and protected from light.

Then wash the cells with PBS five times and store at four degrees Celsius protected from light until they are ready to be imaged in this step. Wash the electroporated cells twice with four degrees Celsius PBS. After four hours of recovery, lice the cells with one milliliter of lysis buffer on ice.

Promptly scrape the cells with a rubber policeman and collect into the conical tubes. Then lice the cells by sonication and vigorous vortexing. Next, centrifuge the sample at 10, 000 times.

Gravity for 10 minutes at four degrees Celsius to collect the cellular debris and insoluble aggregates and save the supernatant. Combine one milliliter of the clarified electroporated cell lysate with 50 microliters of strept in aros resin suspension. Incubate the mixture at four degrees Celsius overnight with end over end rotation to capture the electroporated protein and associated complexes via the strp havein binding peptide affinity tag.

After that, centrifuge the sample at 2, 500 times gravity for two minutes and discard the supernatant. Then wash the sample twice with one milliliter of PBS. Next, add 30 microliters of four XLDS loading buffer, 20 microliters of distilled H2O and one microliter of 0.5 molar TCEP to the sample, and leave some supernatant behind.

Heat the sample at 95 degrees Celsius for 10 minutes. Then cool it on ice for about one minute. Subsequently, centrifuge the sample at 10, 000 times gravity at four degrees Celsius for five minutes.

At the end, collect the supernatant for western blot with appropriate antibodies. Hela cells were incubated or were electroporated with 50 micrograms per milliliter of an affinity tagged salmonella effector, GTGE and stained with an anti effector tag antibody dpi, A nuclear mask, and WGA To delineate the cytosolic boundary incubated hela cells show an absence of green fluorescence indicating a lack of internalization of GTGE. This is a representative photo micrograph of the Electroporated GTG, which shows the intracellular electroporated effector protein shown.

Here is the western blot to detect the eukaryotic interacting partner sine thine protein kinase N one. Note that the far right lane includes two pooled cuvettes. The signal from one qve blended into the background, yet pkn one was still enriched above the lack of binding seen in the no bait control by confocal microscopy after electroporation with 50 micrograms per milliliter.

S sph one hela cells show the colocalization between pkn one and S SPH one. This optical overlap indicates that the effector and the host protein are close enough to physically interact. The fact that the interaction was shown in the nucleus for the first time offers additional support that the protein was trafficked correctly by the host.

Once mastered, this technique can be performed in a high throughput manner. To study multiple individual proteins While attempting this procedure, it's important to remember to empirically determine the best electroporation conditions for each cell type and protein target. This procedure may allow you to multiplex with several proteins, small molecules, or a combination of both to better understand the cellular processes in a variety of model systems.

After watching this video, you should have a good understanding of how electroporation can be used to introduce macromolecules such as proteins into mammalian cells.