The overall goal of the following experiment is to identify interaction partners of grab two by CD NA expression cloning and observation of changes in green fluorescent protein tagged Grab two. This is achieved by first preparing a CD NA library from bacterial lysates as a second step. The CDNAs are transfected in human embryonic kidney cells, which will result in expression of the reporter construct along with the genes of interest.
Next, the transfected plates are analyzed on the opera microscope in order to visualize changes in grab two localization throughout the cells, results are obtained that show epidermal growth factor receptor mediated recruitment of GFP tagged. Grab two to endosomes based on fluorescence microscopy. The main advantage of this technique compared to existing methods such as these two hybrid is that interaction partners of grab two can be identified in a physiological situation that's taking into account the subcellular distribution of the interaction partners.
Well though this method can be used to identify signal transduction by Grab two. It can also be applied to other systems such as other proteins of interest. The CDNA library is provided a single bacterial clones in glycerol stocks.
In a 96 well format using the re ray command in the picker menu of the Hudson Rapid pick light. Pick bacterial clones from the source plate and inoculate into 1.5 milliliters lb amp in a 96 deep well plate. Late seal the deep well plates with a gas permeable seal and incubate overnight at 37 degrees Celsius in a shaker On the following day, spin the deep well plates containing the bacteria A 2000 RPM for 20 minutes.
Using plate adapters in a tabletop centrifuge, aspirate media from the wells leaving the bacterial pellet intact. Next, configure the deck of the Tecan Freedom Evo Automation workstation as shown here. Distribute solutions for plasmid preparation into troughs one to five and place a deep well plate on the deck.
Place a multi-well trough with alucian solution adjacent to the deep well plate load tips into the tip racks. Assemble the vacuum manifold. The plasmid binding plate is placed inside the manifold and the plasmid filter plate is placed on top of the manifold Resus suspend pellets with 250 microliters of solution one.
The Resus suspension buffer by pipetting up and down. Making sure that the pellets are completely resuspended is critical to the success of this procedure. A shaker can be used if a petting up and down does not resuspend the pellet completely.
Once the pellets are completely resuspended lies the bacteria by adding 250 microliters of solution to the lysis buffer. Seal the plate and invert to allow complete mixing. Set a timer for two minutes.
Next, add 350 microliters of solution three. The neutralization buffer. Seal the plate and invert three times lines.
Transfer the bacterial lysates to the plasmid filter plates. Apply vacuum for five minutes. Remove the filter plate and place the binding plate on top of the manifold.
Apply vacuum for one minute. Add 500 microliters of additional wash buffer and apply vacuum for two minutes. Add 900 microliters of wash buffer solution four and apply vacuum for two minutes.
After that, add 900 microliters of wash buffer solution four again and apply vacuum for 15 minutes. Place the DNA collection plate inside the manifold. Add 100 microliters of Elucian buffer to the binding plate and incubate for two minutes.
Apply vacuum for 10 minutes. Finally, remove the collection plate and measure DNA concentration using the NanoDrop 8, 000. Typically a yield of about 100 nanograms per microliter can be expected with this method to begin this procedure.
After hec 2 9 3 cells are tryps, inized and counted. Use an automated dispenser to dispense two times 10 to the four cells into each well of a Perkin Elmer. View plate incubate cells overnight at 37 degrees Celsius and 5%carbon dioxide for transfection.
Use the automated liquid handler to prepare the following mix in each well of a round bottom 96, well plate 100 nanograms of GFP. Grab two plasmid DNA with 100 nanograms of CD NA in 25 microliters of serum free medium. Next, add 25 microliters of serum free media containing 0.5 microliters transfect in per well mix and start the timer for 30 minutes.
After 30 minutes, transfer 50 microliters at the CDNA transfect in mix to cells. Using a gentle dispensing method. Incubate cells overnight at 37 degrees Celsius and 5%carbon dioxide.
To begin image acquisition, start the opera software. Select the configuration tab and select the 20 times objective and the correct plate type. Ensure collar is set to the correct value on the objective to allow for focusing with different plate types.
Next, select the microscope tab. Define exposure one as fite 4 88 laser and exposure two as UV 365 laser. Activate the UV filter on both exposures and assign exposure.
One to camera one and exposure. Two to camera two. Set exposure times to 800 milliseconds for exposure, one and 40 milliseconds for exposure.
Two, select exposure, one set focus height to zero microns. Select focus once focused. Expose camera one.
Adjust the focus height to optimize the exposure plane and click on take height. Save exposure parameters, repeat for exposure. Two, select experiment definition tab, create layout and sub layout.
Drag and drop the relevant layout, exposure, reference image, skew, crop file and sub layout. Save the experiment. Select automatic experiment tab and acquire images under normal conditions.
Grab two is localized throughout the entire cell. Upon stimulation of a growth factor receptor, it translocates to the plasma membrane and is subsequently internalized into endosomes. In this figure, the top panel shows CO M six cells that were transiently transfected with GFP grab two, and as expected, the fluorescence is distributed throughout the cell.
However, in cells transiently transfected with GFP grab two plus the cell surface receptor IT EGFR grab two relocates to endosome like structures as shown in the bottom panel. Thus, when applying a genome wide library, it can be expected that novel grab two binding cell surface proteins can be identified. This method is not limited to the detection of cell surface proteins.
For instance, the GPA's DYNAMIN two, which is involved in grab two mediated endocytosis, induces a translocation of grab two. In this experiment, GFP grab two was cot transfected with DNM two into HEC 2 93 T cells. The relocation of GFP grab two to endosome like structures is illustrated by the cells within the red circles.
Following this procedure, other methods like immunoprecipitation or biochemical assays can be used to answer further questions such as whether the interaction is direct or indirect. After watching this video, you should have a good understanding of how to use expression cloning in order to identify subcellular protein interactions.
