Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

The overall goal of this experiment is to compare the advantages and disadvantages of co-immunoprecipitation, or co-ip, and the proximity ligation assay, or pla, in visualizing protein protein interactions in different cellular compartments. The here presented comparison of two different methods can help scientists to decide which one is better suited for their specific purpose when investigating the distribution patterns of protein protein interactions in the nucleus and the cytoplasm of a cell. The main advantage of co-ip is to detect authentic protein protein interactions.

By contrast, the benefit of pla is to analyze institute protein protein interactions in a rapid way at the single cell level. After growing and harvesting hex293 cells according to the text protocol, to 3 times 10 to the 6th cells, add 300 microliters of cold cytoplasmic extraction solution, and vortex the cells at the highest speed for 15 seconds. Incubate the extract on ice for 30 minutes, and every 10 minutes during the incubation, vortex the sample for five seconds.

Then add 16.5 microliters of cold cytoplasmic extraction solution, vortex for five seconds, and incubate the sample on ice for five minutes. Vortex the extract for five seconds and centrifuge the sample at 16, 000 x g and 4 degrees Celsius for five minutes. Transfer the supernatent into a new prechilled tube and keep it on ice until use, then use one milliliter of cold PBS to wash the insoluble pellets three times by pipetting up and down and removing the PBS after each wash.

Next, add 150 microliters of cold nuclear extraction solution, vortex for 15 seconds, and incubate on ice for one hour. Every 10 minutes during the incubation, vortex the sample for 15 seconds, and use a 200 microliter tip to pipette the solution 10 times. Vortex the tube for 15 seconds, and centrifuge the sample at 16, 000 x g at 4 degrees Celsius for 10 minutes, then transfer the supernatent containing the nuclear extract into a new prechilled tube and keep it on ice until use.

Remove 10%each of the volume of nuclear and cytoplasmic extracts as inputs, then add cold PBS to the remaining extracts to a final volume of one milliliter. Keep on ice until use. After preclearing the protein g conjugated magnetic beads if necessary, combine 40 microliters of protein g conjugated magnetic beads with four micrograms of rapid polyclonal anti-RBM3 antibody, or rapid IGG as a negative control in 200 microliters of PBST.

Incubate up to room temperature on a rotater for 40 minutes. Place the tubes on a magnetic rack, then remove and discard the supernatent. Then with 200 microliters of PBST, wash the beads once.

Add one milliliter of diluted lisates to the beads, and incubate on a rotator at four degrees Celsius overnight. The following day, place the tubes on the magnetic rack and aspirate the supernatent. Add 0.5 milliliters of PBST to the beads, and wash them three times on a rotator at four degrees Celsius, with a fixed speed of 20 rpm for 10 minutes.

Elute the protein from the beads by adding 40 microliters of sample buffer, then heat the sample at 70 degrees Celsius for 10 minutes. After spinning down the beads, transfer the liquid to a new 1.5 milliliter tube. To carry out immunocytic chemistry, seed HEK293 cells at 1.5 times 10 to the four cells per chamber in an eight chamber polydelysine coated slide.

In 0.4 milliliters of DMEM supplemented with 10%FPS and 100 units per milliliter of penstrep. Incubate the cells at 37 degrees Celsius and 5%carbon dioxide for 48 hours, then aspirate the medium and use 4%PFA to fix the cells at room temperature for 10 minutes in a laminar flow hood. Then aspirate the PFA and use 0.5 milliliters of PBS per chamber to wash the cells three times.

Add 0.5%tritonX100 with 5%NGS and PBS to permeablize and block the cells and incubate at room temperature for one hour. Then, after diluting anti NF90 monoclonal and anti RBM3 polyclonal primary antibodies one to 100, add primary antibodies to the wells and incubate on the shaker at four degrees Celsius overnight. The following day, use 0.5 milliliters of PBS to wash the chambers three times for 10 minutes each.

To carry out immunocytic chemistry, add a one to 500 dilution of green fluorescent dye coupled antimouse and red fluorescent dye coupled antirabbit antibodies to the cells, and incubate at room temperature for one hour. Add a one to 5, 000 dilution of DAPHE NBPS to counterstain the nuclei and incubate at room temperature for 10 minutes, then wash the samples with PBS as before. Remove the chambers from the glass slide and dry them, Before using 250 microliters of mounting medium per slide to mount them.

To prepare tube proximity ligation assay probes, after incubating cells and primary antibodies, and washing as just demonstrated, dilute the premade probes one to 5 in 0.1%tritonx100 and 5%NGS and PBS, in a total volume of 320 micorliters for one eight chamber slide. Incubate the solution at room temperature for 20 minutes. Remove the chambers from the glass side and add the diluted probes, then incubate the chambers in a humidity incubator at 37 degrees Celsius for one hour.

Prepare a ligation solution by mixing eight microliters of ligase, 64 microliters of 5x ligation stock, and 248 microliters of water. Tap off the liquid from the slide, and use onex wash buffer A to wash the samples twice for five minutes each. Add the ligation solution to the samples, and incubate them in a humidity incubator at 37 degrees Celsius for 30 minutes.

Prepare the amplification solution by mixing four microliters of polymerase, 64 microliters of 5x amplification stock, and 252 microliters of water, and wrap the tube in foil to protect it from light. Then, tap off the liquid from the slide, and use one x wash buffer A to wash the samples twice for two minutes each. Add the amplification solution to the samples and incubate at 37 degrees Celsius in a dark humid incubator for 100 minutes.

Then after tapping off the liquid as before, wash the samples twice for 10 minutes each in wash buffer B before washing them once in 0.1 x wash buffer B.Finally, dry the slide and mount the samples with 250 microliters of commercial mounting medium with DAPHE, examine the flourescence under a microscope, and acquire images with CCD camera. Co-immunoprecipitation experiments with RBM3 as the bait protein revealed that NF90-RBM3 interactions are predominantly present in the nucleus and a minority are found in the cytoplasm. In this immunoflourescence experiment, NF90 and RBM3 are both seen in the nucleus.

Both proteins are also found in the cytoplasm in C2, and both proteins show perfect colocalization in each compartment. As seen in these images, the proximity ligation assay revealed the pattern of NF90-RBM3 interactions, which is very similar to convention immunocytic chemisty, which most interactions in the nucleus in the majority of cells. Only a small proportion of cells demonstrated predominantly cytoplasmic distribution of NF90-RBM3 interactions.

While attempting this procedure, it's important to remember that, although more convenient, PLA does not reflect protein protein interactions directly. Instead co-ip detects directed interaction. In addition, the specificity of the primary antibody determines the quality of the result.

After watching this video, you should have a good understanding of how to choose an appropriate method to study protein protein interactions in different cellular compartments according to your own experimental purpose.