Proximity Ligation Assay to Study In Situ Protein-Protein Interactions

Begin the proximity ligation assay by taking dissected nematodes and fixing them on a slide with their germline cells exposed. These cells exhibit high levels of ribonucleoprotein complexes containing RNA-binding proteins, RBPs, associated with their partner proteins.

Overlay the slide with two primary antibodies that recognize two specific target proteins — the RBP and its partner protein. Supplement the slide with two types of secondary antibodies specific to their respective primary antibodies.

Each secondary antibody is conjugated with PLA probes — DNA strands with complementary sequences. The secondary antibodies bind to the pre-attached primary antibodies, bringing the two PLA probes close.

Apply the ligation buffer containing connector oligonucleotides — which hybridize with PLA probes — and ligase enzyme seals the gap, generating circular DNA. Incubate the slide with an amplification solution containing DNA polymerase, dNTPs, and detector probes attached to a red fluorophore.

Using the circularized DNA as a template, the polymerase initiates a rolling circle amplification, generating many copies of the template DNA connected to each other. The amplified DNA remains tethered to the PLA probe, enabling protein-protein interaction localization.

The detector probes, carrying short single-stranded oligonucleotides, interact with the complementary regions of the amplified DNA, forming duplexes and imparting fluorescence signals to protein complexes. Image the slide under a fluorescence microscope.

Observe for distinct red spots inside the cell, indicating successful protein-protein interactions.

Use the antibody diluent to dilute the primary antibodies. Apply 40 microliters of the diluted primary antibody solution per 14 by 14-millimeter space on the slides. Place the slides in a humid chamber, and incubate overnight at 4 degrees Celsius.

In the morning, wash the slides twice for 5 minutes each with 50 milliliters of 1x wash buffer A at room temperature in a Coplin jar. Set the Coplin jar on an orbital shaker set to 60 RPM. Place the slides on a paper towel to let wash buffer run off the slide, and gently wipe the edges.

Prepare a 40-microliter solution containing plus and minus probes. Add the solution to each 14 by 14-millimeter space. Incubate the slides in a humid chamber for 1 hour at 37 degrees Celsius. Wash the slides twice for 5 minutes each with 50 milliliters of 1x wash buffer A at room temperature in a Coplin jar.

Set the Coplin jar on an orbital shaker set to 60 RPM. Dilute the ligation buffer 1 to 5 with ultrapure water. Use this buffer to dilute the ligase 1 to 40 to prepare a working stock of ligation solution. Place the slide on a paper towel to let the wash buffer run off the sides, and gently wipe the edges.

Add 40 microliters of the ligation solution to each 14 by 14-millimeter space. Incubate the slides in a humid chamber for 30 minutes at 37 degrees Celsius. After washing and drying the slides as previously described, add 40 microliters of freshly-prepared amplification solution protected from light to each 14 by 14-millimeter space.

Incubate the slides in the humid dark chamber for 1 hour and 40 minutes at 37 degrees Celsius. Wash the slides twice for 10 minutes each with 50 milliliters of 1x wash buffer B, and wash the slides one time for 1 minute with 50 milliliters of 0.01x wash buffer B.

After drawing the epoxy-coated perimeter of the slide, add 10 microliters of mounting medium to each sample, and gently lay a coverslip on top, allowing for the mounting medium to spread out. Paint around the edge of the coverslip with nail polish to seal the coverslip and slide. Avoid moving the coverslip. Let the nail polish harden for at least 20 minutes at room temperature protected from light.