Pla probe minus

The analysis was carried out according to the manufacturer’s instructions [34 (link)]. This assay is used to detect, visualize and quantify protein expression, protein interactions and specific post-translational protein modification [34 (link)]. In the cases of detecting protein interactions and post-translational modification, two primary antibodies are required which are raised from different species. The secondary antibodies called PLA probes (PLA probe MINUS and PLA probe PLUS) and each one is conjugated with a unique oligonucleotide, were obtained from Sigma-Aldrich (DUO92008 SIGMA; POOLE UK). When the two PLA probes are in close proximity (<40 nm), the oligonucleotides will hybridize and join to a closed circle by adding enzymatic ligation solution. The ligated circle as a template, is then amplified through rolling circle amplification and generating several-hundredfold repeated sequence product subsequently. Since the amplification solution consists of nucleotides, polymerase as well as fluorescently labelled oligonucleotides, the replicated product can be easily visible as distinct fluorescent spot when viewed with a fluorescence microscopy.

Adherent HeLa cells on the glass slides were transfected with the indicated plasmids. After fixation, cells were pretreated and blocked with 5% BSA. Primary antibodies against GFP and Syk were used for the slides. The secondary antibodies, which were conjugated with oligonucleotides (PLA probe MINUS [Sigma no. DUO92004] and PLA probe PLUS [Sigma no. DUO92002]), were added to the glass slides for 1 h at 37°C. After incubation, a system consisting of two oligonucleotides and a ligase were added to the system for 30 min at 37°C. If the oligonucleotide probes were in close proximity, the ligation reaction between them formed a closed DNA circle. Then, the slides were incubated for amplification by the PLA probe (Sigma no. DUO92008) and the polymerase for 100 min at 37°C. The amplification reaction results in a signal which was seen as a unique fluorescent spot under confocal microscopy. The PLA dots were counted via Fiji software and analyzed as previously described (40 (link)).

Adherent HeLa cells on the glass slides were transfected with the indicated plasmids. After fixation, cells were pretreated and blocked with 5% BSA. The requirements of the primary antibodies were used for the slides. The secondary antibodies, which were conjugated with oligonucleotides (PLA probe MINUS, SIGMA DUO92004 and PLA probe PLUS, SIMGA DUO92002), were added to the glass slides for 1 h at 37 °C. After incubation, a system consisting of two oligonucleotides and a ligase were added to the system for 30 min at 37 °C. If the oligonucleotide probes were in close proximity, they formed a closed circle. Then, the slides were incubated for amplification by the PLA probe (SIGMA DUO92008) and the polymerase for 100 min at 37 °C. The signal was seen as a unique fluorescent spot under confocal microscopy.