Detection of Protein-Protein Interactions in Drosophila Larvae Using a Proximity Ligation Assay

Take the body wall of Drosophila larvae, consisting of muscles innervated by motor neurons to form neuromuscular junctions or NMJs.

The postsynaptic muscle membrane at the NMJ contains interacting membrane-bound proteins, which are labeled with primary antibodies.

Add proximity ligation assay, or PLA, probes containing secondary antibodies conjugated to single-stranded oligonucleotides. Incubate for the probes to bind to the primary antibodies.

Wash away any unbound probes.

Incubate with ligation buffer containing connector oligonucleotides and ligase enzymes.

The proximity of the interacting proteins facilitates connector binding to probes. Ligase then seals the gaps, creating circular DNA.

Wash away any unbound connectors.

Incubate with an amplification solution containing polymerase, which amplifies the circular DNA through rolling circle amplification.

Fluorophore-tagged detector probes in the solution anneal to the amplified DNA, and upon excitation produce a fluorescent signal.

Mount the body wall on a slide to visualize fluorescently labeled protein-protein interactions at the NMJ.