Proximity Ligation Assay for Protein Modification

Protocol Overview

PLA was performed in prostate cancer cells, which were grown on cover slip for 72 h. The cells were fixed in 4% paraformaldehyde/PBS at r. t. for 30 min, washed thrice with PBS and blocked for 1 h in 3% BSA. After treatment with mouse anti-carbohydrate abs and rabbit anti-mucin Abs (1:100 in PBS with 3% BSA) at 37 °C for 1 h, cells were washed thrice (5 min each) with PBST. Oligonucleotide-conjugated anti-mouse minus and anti-rabbit plus PLA secondary probes were added at appropriate dilutions prepared in PBS with 3% BSA and the cells were incubated in a humidified chamber for 1 h at 37 °C. The PLA assay was performed using the PLA kit according to the manufacture’s instruction. Briefly, connector oligonucleotides were hybridized and circularized by ligation for 30 min at 37 °C. After thorough washing, the cells were incubated with DNA polymerase for 100 min at 37 °C to produce rolling circle amplification products tagged with a red fluorescence probe. The nuclei were counterstained with DAPI, and the PLA signals were visualized at 60 x magnifications under Zeiss 510 Meta Confocal Laser Scanning Microscope equipped with DAPI/Texas Red filters and analyzed using Zeiss 510 software.