Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

The overall goal of this procedure is to identify the protein interaction partners of a protein of interest using the DI hydro folate reductase protein fragment COMPLEMENTATION assay, or D-H-F-R-P-C-A. This is accomplished by first condensing the DH FFR F three collection or praise on high density colony arrays. Next, the bait strain is made with the array of praise on rich medium.

In the final step, the diploids resulting from these matings are selected. Ultimately, the reconstitution of the DHFR is quantified by measuring the colony sizes on selective PCA medium, which gives a signal proportional to the amount of bait prey complex reconstituted in the cells. The implications of this technique extend toward the therapy of diseases such as cancer, as it is by the rewiring of protein interaction networks.

That mutations can lead to such diseases. On the morning of the procedure, sterilize the robotic platform by first soaking the PIN tools five times for 10 seconds in the water bath station to remove any residual cell clumps. Then soak the PIN tool twice back and forth in the brush station and twice in the so cater station for 20 seconds each soak to remove any of the remaining cells while the PIN tools are being cleaned.

Turn on the UV lamp for five minutes to sterilize a robot. Enclosure then after the last wash, dry the PIN tools in the air dryer station for 25 seconds. Condense A-D-H-F-R collection onto 16 arrays of 384 strains here.

A 384 array can be subdivided in four equally interest spaced quadrants, each consisting of 96 positions in a two by two matrix layout for a total of four quadrants. To do this for each 384 array, print four glycerol plates on the four quadrants of an omni tray containing YPD plus 250 micrograms per milliliter. Hygromycin B, using the 96 pin tool to do this, insert 4 96 well plates containing the LDH FFR F three negative control in between the 60 glycerol plates from the DHFR three collection in order to have a final set of 64 plates that exactly fill four 1, 536 arrays.

Then insert 4 96 well plates containing the LDH FFR F three negative control in between the 60 other plates to have a final set of 64 plates that exactly fill 4 15 36 arrays before adding cells to the source plates wet the sterilized pins in 35 milliliters of sterile water in an omni tray in the wet station. Incubate the arrays at 37 degrees Celsius for two days, and then to condense the collection into four arrays of 1, 536 strains. Print four arrays of 384 strains on each of the four destination plates.

Using the 384 pin tool. Sterilizing the PIN tool between each replication cycle as just demonstrated after another two day incubation. Standardize the colony size by replicating the four arrays on the selected YPD medium with a 1, 536 pin tool and incubate the plates for 48 more hours.

For high throughput. D-H-F-R-P-C-A first inoculate a culture of the bait strain in 20 milliliters of liquid YPD plus CIO ricin in a 50 milliliter tube and incubate the cells for two days at 30 degrees Celsius with shaking at 250 RPM. When the culture has reached saturation.

Plate five milliliters of the cell suspension onto a YPD plus gnat omni tray, and let the cells absorb on the surface after five to 10 minutes, remove the excess liquid and incubate the culture at 30 degrees Celsius after two days. Use the 1, 536 pin tool to print the bait strain on 12 YPD plates using each cell lawn no more than four times. Then using the 1, 536 pin tool, again, print the appropriate array for the DHFR F three collection on top of the bait cells.

Let the strains mate during another two day incubation and then select the diploid cells by printing the colonies on omni trays containing YPD plus hydro mycin B and nortin. Incubate the diploids for two more days at 30 degrees Celsius, and then repeat the selection as just demonstrated one day into the incubation. Pour plates with media containing methotrexate the next day.

Use the 1, 536 pin tool to print diploid cells onto the methotrexate medium and incubate the plates for four more days in plastic bags to keep the cultures from drying out. On the third day of the incubation. Pour a second batch of omni trays containing MTX medium as just demonstrated the next day, turn on the robot light and use a robotic platform to image the plates to decrease the background growth of the PCA strains and to increase the quantitative resolution, replicate the cells on the second batch of MTX media to perform a second round of MTX selection as just demonstrated.

Finally, after acquiring images from the second set of plates, analyze the colony arrays with the appropriate software, collecting the colony size information for each position of each array. The threshold defined with the LDH FFR F three controls can be used as an empirical threshold to determine the high confidence hits. The known physical interactors of the bait can then be retrieved from databases like Biogrid and overlaid on the data.

For example, here, five out of eight high confidence hits were previously reported as NUP 82 interactors, amongst which others NUP one 16 and NUP 1 59 were determined to be part of the NUP 82 sub complex. The data also indicate that PEX 30 approximal membrane protein may represent a novel physical interactive of Nup 82 as confirmed using D-H-F-R-P-C-A at a low throughput two of the other interaction partners detected. New one 20 and new 85 were determined to not be part of the new 82 sub complex illustrating the ability of D-H-F-R-P-C-A to detect the interactions within and between sub complexes of larger complexes.

While attempting this procedure, it's important to remember to use Freshly Report media to avoid any troubleshooting with the printing steps, and to include the necessary controls to, to make sure that the final PCA media enables growth of only cells demonstrating DHFR complementation.