Proximity Ligation Assay to Study In Situ Protein-Protein Interactions
Transcrição
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.
